Engineering pathogen resistance in plants

ABSTRACT

Provided herein are compounds and methods for increasing disease resistance and/or root length in plants.

This application is a continuation application of U.S. patent application Ser. No. 15/382,461, filed Dec. 16, 2016, now U.S. Pat. No. 10,407,691, issued Sep. 10, 2019, which is a continuation-in-part of PCT Patent Application No. PCT/US2015/036439, filed Jun. 18, 2015, which claims benefit of priority to U.S. Provisional Patent Application No. 62/013,709, filed Jun. 18, 2014, which are all incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The invention was made with government support under Contract Nos. DE-AC02-05CH11231 awarded by the U.S. Department of Energy, and Grant No. R01 GM59962 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Perception of extracellular signals by cell surface receptors is of central importance to eukaryotic development and immunity. Cell surface Pattern Recognition Receptors (PRRs), play an essential role in the innate immune responses in animals and plants. PRRs share conserved signaling domains, such as leucine-rich repeats (LRRs), and function via kinases, which are either integral to the receptor (plants) or associated with the receptor (animals). Many of these receptors regulate transcription of target genes through phosphorylation events after recognition of pathogen associated molecular patterns (PAMPs).

In animals, recognition of PAMPs in extracellular compartments or at the cell surface is largely carried out by the toll like receptor (TLR) family that contains LRRs in the extracellular domain and a TIR intracellular domain. All TLRs activate a common signaling pathway to induce a core set of defense responses via kinases. TLRs associate with the interleukin-1 receptor-associated kinase (IRAK) family and with receptor interacting-protein (RIP) kinases via adaptor proteins. In plants, cell surface recognition of PAMPS is largely carried out by receptor kinases (RKs).

Kinases can be divided into “RD” kinases, which have an arginine (R) immediately preceding the conserved catalytic aspartate (D), and a smaller group of “non-RD” kinases, which typically carry a cysteine or glycine before the aspartate residue. The RD class of kinases includes nearly all receptor tyrosine kinases (RTKs) and most characterized plant RSTKs. The non-RD class includes members of human IRAKs and RIPs, Drosophila Pelle, and members of plant RKs belonging to the IRAK family.

Plant genome analyses have revealed the presence of a large family of these non-RD IRAK RKs, with more than 45 encoded in the Arabidopsis genome and more than 370 found in the rice genome. Members include Arabidopsis FLS2 that detects the peptide flg22, a component of flagellin; the elongation factor Tu receptor (EFR) that detects elf18, a peptide corresponding to the bacterial elongation factor Tu; the rice XA26 and Pi-d2 RKs; and the rice XA21 RK that mediates recognition of Xanthomonas oryzae pv. oryzae (Xoo).

Activation of XA21 results in a robust immune response. XA21 biogenesis takes place in the endoplasmic reticulum. After processing and transit to the plasma membrane, XA21 binds to XB24 (XA21 binding protein 24). XB24 associates with the XA21 juxtamembrane domain and catalyzes the autophosphorylation of serine and threonine residues on XA21, maintaining XA21 in an inactive state. Following pathogen recognition, the XA21 kinase disassociates from XB24 and is activated. XA21 also binds to XB25, a plant-specific ankyrin-repeat protein. XB25 associates with the XA21 transmembrane domain in vitro and is weakly transphosphorylated by XA21. The XA21-mediated signaling pathway is attenuated by XB15, a protein phosphatase 2C, which dephosphorylates XA21. XA21-mediated immunity requires the presence of a co-receptor called OsSERK2 (somatic embryogenesis receptor kinase 2). OsSERK2 interacts with the intracellular domain of XA21 in a kinase activity dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo.

BRIEF SUMMARY OF THE INVENTION

The present results reveal a microbial molecule that activates XA21-mediated immunity (sulfated raxX), and molecular pathways involved in its processing (e.g., raxST) and release (e.g., membrane fusion protein (MFP) (e.g., raxA), ABC transporter protein (e.g., raxB and/or cvaB) and outer membrane protein (OMP) (e.g., raxC)).

Provided herein are isolated or purified or synthetic RaxX polypeptides or homologs thereof. In some embodiments, the polypeptide comprises a sequence of SEQ ID NO:46, or a sequence having at least 60% identity (e.g., at least 65%, 70%, 75%, 80%, 85%, or 90%) to SEQ ID NO:46, wherein the amino acid at the position corresponding to Tyr2 of SEQ ID NO:46—which corresponds to Tyr10 of SEQ ID NO:1—(i.e., when the sequences are optimally aligned) is Tyr. In some embodiments, the polypeptide comprises the sequence DY(P/G)(P/D)P(G/S)ANPKHDP, wherein the amino acids in parentheses are options at that position. In some embodiments, the peptide is not DYGDPSANPKHDP (SEQ ID NO:47). In some embodiments, the polpeptide comprises any of the 13mer sequences depicted in FIG. 45 part 2 from the highlighted “D” through the highlighted “P” or an aligned sequence thereof in the Figure. In some embodiments, the polpeptide comprises a sequence from Table 51.

In some embodiments, the polypeptide comprises a sequence of SEQ ID NO:1, or a sequence having at least 60% identity (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, or 95%) to SEQ ID NO:1, wherein the amino acid at the position corresponding to Tyr10 of SEQ ID NO:1 (i.e., when the sequences are optimally aligned) is Tyr.

In some embodiments, the polypeptide as described above or elsewhere herein is 13, 14, 15, 16, 17, 18, 19, 20, 13-20, 13-75, 15-75, 20-50, 50-100, 50-75, or about 60 amino acids in length. In some embodiments, the Tyr corresponding to Tyr10 of SEQ ID NO:1 is sulfated. In some embodiments, the polypeptide comprises a sequence of any one of SEQ ID NOs:22-45, or amino acids 20-60, 25-55, or 30-55 of any one of SEQ ID NOs:22-45. In some embodiments, the polypeptide comprises amino acids 32-51 of SEQ ID NO:22. In some embodiments, the polypeptide is encoded by a polynucleotide selected from the group of sequences consisting of SEQ ID NOs:5-21.

Further provided are agricultural compositions comprising the polypeptide described above formulated for application to a plant (e.g., directly, or via soil or water). In some embodiments, the agricultural composition further comprises an agriculturally suitable carrier, surfactant, herbicide, fungicide, pesticide, or fertilizer. In some embodiments, the polypeptide or agricultural composition enhances the disease resistance of a plant expressing XA21 or a homolog thereof (endogenously or heterologously expressed) when contacted with the plant, compared to a plant that is not contacted with the polypeptide or agricultural composition. In some embodients, the tyrosine corresponding to Tyr10 of SEQ ID NO:1 or Tyr2 of SEQ ID NO: 46, is sulfated. In some embodiments, the polypeptide is 13, 14, 15, 16, 17, 18, 19, 20, 13-20, 13-75, 15-75, 20-50, 50-100, 50-75, or about 60 amino acids in length.

Additionally provided are plants contacted with the polypeptide or agricultural composition as described above. In some embodiments, the plant expresses XA21 or a homolog thereof (endogenously or heterologously). In some embodiments, the plant has enhanced disease resistance compared to a plant not contacted with the polypeptide or agricultural composition. In some embodiments, the plant is of a species selected from the group consisting of: Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, eucalyptus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Setaria, Miscanthus, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.

Provided herein are methods of making the polypeptide as described above comprising introducing an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding the polypeptide to a heterologous cell, expressing the polypeptide in the cell, and purifying the polypeptide. In some embodiments, the promoter is a pathogen-inducible promoter. In some embodiments, the polypeptide comprises a cleavable tag to ease purification, e.g., a maltose binding protein (MBP) or poly-His tag. After the expressed polypeptide is separated from extraneous material, the tag can be removed enzymatically or chemically according to common techniques.

In some embodiments, the polynucleotide sequence encoding the polypeptide comprises an amber codon at the amino acid position corresponding to Tyr10 of SEQ ID NO:1, and the heterologous cell comprises an orthogonal aminoacyl-tRNA synthetase/tRNA pair, and sulfotyrosine. In some embodiments, the heterologous cell comprises an enzyme that sulfates the Tyr at the position corresponding to Tyr10 of SEQ ID NO:1. In some embodiments, the method further comprises introducing to the cell an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding the enzyme. In some embodiments, this orthogonal expression system is carried out in bacteria, e.g., E. coli.

Further provided are methods of making the polypeptides as described herein using chemical synthesis. The length of the raxX polypeptide required to activate XA21-mediated immunity is small (e.g., about 20 amino acids), making chemical synthesis an economically viable option. In some embodiments, the method comprises adding a sulfated tyrosine to the nascent polypeptide chain at the amino acid position corresponding to Tyr10 of SEQ ID NO:1.

Also provided are transgenic plants, wherein the plant comprises a heterologous expression cassette comprising a promoter (e.g., a pathogen-inducible promoter) operably linked to a polynucleotide encoding a polypeptide as described above. In some embodiments, the plant expresses endogenous XA21 or a homolog thereof. In some embodiments, the plant further comprises a heterologous expression cassette comprising a promoter operably linked to a polynucleotide encoding XA21 or a homolog thereof. In some embodiments, the plant further comprises a heterologous expression cassette comprising a promoter operably linked to a polynucleotide encoding a sulfotransferase (e.g., RaxST or a homolog thereof). In some embodiments, one, two, or three of the promoters are pathogen-inducible promoters. In some embodiments, the plant is of a species selected from the group consisting of: Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, eucalyptus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Setaria, Miscanthus, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.

Provided herein are methods for enhancing disease resistance and/or enhancing root growth in a plant. In some embodiments, the method comprises contacting the plant with a sufficient amount of the polypeptide or the agricultural composition as described above to enhance the disease resistance and/or root growth of the plant compared to a plant that is not contacted by the polypeptide or agricultural composition. In some embodiments, the plant expresses XA21 or a homolog thereof (endogenously or heterologously). In some embodiments, the plant is of a species selected from the group consisting of: Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, eucalyptus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Setaria, Miscanthus, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea. In some embodiments, the method further comprises contacting the plant with a pathogen (e.g., Xanthomonas bacteria, a non-Xanthomonas pathogen, or supernatants or extracts from the pathogen) and determining disease resistance to the pathogen.

Also provided are recombinant host cells, wherein the host cell comprises a heterologous expression cassette comprising a promoter operably linked to a polynucleotide encoding a RaxX polypeptide or homolog thereof as described above. In some embodiments, the host cell is selected from the group consisting of a plant cell, a fungal cell, a bacterial cell, a yeast cell, an insect cell and a mammalian cell. In some embodiments, the host cell expresses (endogenously or heterologously) an enzyme that sulfates the Tyr corresponding to Tyr10 in SEQ ID NO:1 (if present), e.g., RaxST or a homolog thereof (e.g., having an Arg at the amino acid position corresponding to Arg35 in Xoo RaxST). In some embodiments, the host cell expresses (endogenously or heterologously) a membrane fusion protein, ATP binding cassette transporter protein, and outer membrane protein, e.g., RaxA, RaxB, and RaxC, or homologs thereof.

Further provided is a method of screening for a polypeptide that enhances disease resistance (e.g., XA21-mediated disease resistance or resistance mediated by a XA21 homolog) and/or root growth in a plant. In some embodiments, the method comprises a) detecting polypeptides released from a bacterial cell lacking functional raxB and cvaB (raxB/cvaB deficient); b) detecting polypeptides secreted from a bacterial cell having functional raxb and cvaB; c) identifying polypeptides released in b) that are not released in a); d) applying a polypeptide identified in c) to the plant; and e) selecting the polypeptide that enhances disease resistance compared to a plant not contacted with the polypeptide. In some embodiments, the plant expresses XA21 or a homolog thereof (endogenously or heterologously). In some embodiments, the method further comprises contacting the plant with a pathogen, e.g., a Gram negative bacteria such as Xanthomonas, or supernatants or extracts from the bacteria.

Also provided are methods of screening for compounds that affect expression of Rax genes. In some embodiments, the method comprises (i) contacting a test compound with a cell comprising an expression cassette having a Rax gene promoter and either a Rax-encoding sequence, or a reporter-encoding sequence (e.g., GFP); (ii) detecting the level of Rax expression by detecting the level of Rax gene product (e.g., RNA or protein) or reporter; and (iii) comparing the level detected in step (ii) to the level of Rax expression in a control cell. In some embodiments, the control cell is not contacted with the test compound. In some embodiments, the Rax gene is RaxX. In some embodiments, the Rax gene is RaxST. In some embodiments, the expression cassette is an endogenous gene in the cell (e.g., a Gram-negative bacterial cell). In some embodiments, the expression cassette is heterologous to the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Model for the synthesis and secretion of proteins required for activation of XA21-mediated immunity (encoded by rax genes). In this model, the adenosine-5′-triphosphate (ATP) sulfurylase RaxP and the adenosine-5′-phosphosulfate (APS) kinase RaxQ catalyze the production of the universal sulfuryl group donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The RaxST sulfotransferase utilizes PAPS as a substrate to sulfate RaxX. RaxX contains a leader sequence (L), which we hypothesize is cleaved by the peptidase domain of RaxB and secreted outside the bacterial cell by the RaxABC T1SS. CvaA and CvaB, which are predicted orthologs of RaxA and RaxB, respectively, are hypothesized to serve as an alternate T1SS to process and secrete sulfated RaxX.

FIG. 2. raxST is required for activation of XA21-mediated immunity. The standard leaf clipping method (Jiang et al. (2013) Plant J. 73:814) was used to inoculate rice plants (TP309 and TP309-XA21) with a 10⁸ CFU/mL bacterial suspension. Lesion lengths were measured 14 days post inoculation. Bars represent the mean of at least 10 leaves±SE.

The following Xoo strains were used: PXO99A WT [PXO99A], PXO99A ΔraxST [RaxST], PXO99A ΔraxST complemented with RaxST [RaxST (RaxST)], and PXO99A ΔraxST complemented with catalytic inactive RaxST (R35a) [RaxST (STr35a)].

FIG. 3. The raxB/cvaB double mutant overcomes XA21-mediated immunity. The standard clipping method was used to inoculate rice plants (TP309 and TP309-XA21) with a 10⁸ CFU/mL suspension. Lesion lengths were measured 14 days post inoculation. Bars represent mean of 20-30 leaves±SE. Statistical significance was calculated using the Tukey-Kramer HSD test. Different letters indicate significant difference (p<0.01). This experiment was repeated 3 times with similar results.

The following Xoo strains were used: PXO99A ΔraxST [ΔraxST], PXO99A WT [PXO99A], PXO99A ΔraxB [raxB], PXO99A ΔraxB/AcvaB [raxB/cvaB] and PXO99A ΔcvaB [cvaB].

FIG. 4. raxX is required for activation of XA21-mediated immunity. The standard clipping method was used to inoculate rice plants (TP309 and TP309-XA21) with a 10⁸ CFU/mL bacterial suspension. Lesion lengths were measured 14 days post inoculation. Bars represent mean of at least 10 leaves±SE. The experiment was repeated 3 times with similar results.

The following Xoo strains were used: PXO99A WT [PXO99A], PXO99A ΔraxST [RaxST], PXO99A carrying a deletion of 1 kb located 300 bp upstream of the start site of RaxST [Ups], PXO99A Ups complemented with the open reading frame of RaxX [Ups(RaxX)] and PXO99A Ups complemented with the open reading frame of RaxX with a mutation in the presumed start codon (atg-ccc) [Ups(RaxX-atg-ccc)] and PXO99A Ups complemented with the open reading frame of RaxX(Y41F) [Ups(RaxX-Y41F)].

FIGS. 5A and 5B. Expression and purification of RaxX-His and RaxX(sY)-His. A specifically engineered orthogonal aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pair was co-expressed from the pULTRA_SYP29 plasmid. This tRNA recognizes the amber codon and incorporates sulfotyrosine, which is supplied in the media, at the desired position MBP-3C-RaxX-His or MBP-3C-RaxX(sY)-His were expressed in pMAL vector in E. coli BL21 cells in the presence of 1 mM IPTG for 16 h at 20° C. Both proteins were two-step purified and RaxX-His released from the N-terminal MBP-tag by 3C protease treatment. (FIG. 5A) Sulfated RaxX(sY)-His (+SO₄ ²⁻) runs at a slightly higher molecular weight than RaxX-His most likely due to its additional negative charge. (FIG. 5B) Sulfation of the central tyrosine of RaxX can only be detected on trypsin peptides of RaxX(sY)-His (lower panel) but not of RaxX-His. SEQ ID NO:4 is the 15-mer peptide used in this test.

FIG. 6. RaxX-His induces defense gene expression in a tyrosine sulfation and XA21-dependent manner. Leaves of Kitaake or Kitaake expressing Ubi::XA21::MYC were treated with about 200 nM purified mock, RaxX-His (designated RaxX-Y) and RaxX(sY)-His) designated RaxX-Y-S) for 24 h. PR10b marker expression was measured by qPCR, and normalized to actin expression. Defense gene expression is only induced in XA21 plants in the presence of sulfated RaxX but not nonsulfated RaxX. Similar gene expression patterns were observed for Os04g10010. The experiment was repeated 8 times with similar results.

FIGS. 7A-7C: Amino acids 32-55 of Xoo RaxX-Y are sufficient for activating XA21-mediated gene expression. Amino acids 21-60 of RaxX (SEQ ID NO:3) were digested with the indicated proteases as shown in FIG. 7C. Leaves of Kitaake expressing Ubi::XA21::MYC were treated with about 500 nM or 1 uM of the resulting peptides. Os06g13390 marker expression was measured by qPCR. FIG. 7A shows results for 500 nM and FIG. 7B shows results for 1 uM.

FIG. 8 shows the percent identity of amino acid sequences of RaxX proteins for the indicated strains.

FIG. 9 shows the percent identity of nucleic acid sequence encoding RaxX proteins for the indicated strains.

FIGS. 10A and 10B show an alignment of RaxX polypeptide sequences from various Gram negative bacteria. The amino acid sequences are designated SEQ ID NO:22 (consensus sequence) to SEQ ID NO:45.

FIGS. 11A to 11D. raxX, a small ORF located upstream of the raxSTAB operon, is required for activation of XA21-mediated immunity. (FIG. 11A). raxST, raxA, and raxB are encoded in a single operon. A 1.0 kb region upstream of raxST and a 1.7 kb region downstream of raxB were deleted in PXO99Δ1.0^(Sp) and PXO99Δ1.7^(Sp), respectively. The raxX ORF is located ˜0.4 kb upstream of raxST and in the opposite orientation. (FIG. 11B) TP309 (open bars) or XA21-TP309 (black bars) were inoculated by clipping with scissors dipped in Xoo suspensions. Bars indicate the mean lesion length±standard error (SE) measured 14 days after inoculation (n ≥14). The ‘*’ indicates statistically significant difference from PXO99 using Dunnett's test (α=0.01). No statistical differences in lesion length were observed on TP309 inoculated with the different strains. The experiment was repeated at least 3 times with similar results. (FIG. 11C) XA21-TP309 rice leaves display water soaked lesions two weeks after inoculation with the indicated strains. (FIG. 11D) Growth of PXO99 (□), PXO99ΔraxX (Δ), and PXO99ΔraxX(praxX) (⊖) in rice leaves inoculated as in FIG. 11B. In planta bacterial growth analysis was carried out as described (Bahar O, et al. (2014) PeerJ 2:e242). Bacterial quantification was determined as the number of colony forming units (CFU) extracted per inoculated leaf. For the final data point, ‘*’ indicates statistically significant difference from PXO99 using Dunnett's test (α=0.01, n=4). The experiment was repeated twice with similar results.

FIGS. 12A-12D. RaxST sulfates RaxX on tyrosine 41. (FIG. 12A) The predicted PAPS binding residue R35 of RaxST and Y41 of RaxX are required for activation of XA21-mediated immunity. TP309 (open bars) and XA21-TP309 (black bars) were inoculated with the indicated Xoo strains, and lesion lengths were measured 14 days later as described in FIG. 11B. Bars indicate the mean lesion length±SE (n≥14). The ‘*’ indicates statistically significant difference from PXO99 using Dunnett's test (α=0.01). (FIG. 12B) UV photodissociation (UVPD) mass spectrum of a tyrosine sulfated peptide (HVGGGDsYPPPGANPK (SEQ ID NO:4)), 2-, m/z 770) from trypsin digestion of in vitro sulfated Rax39. After incubation with E. coli-expressed and purified His-RaxST, RaxX39 was digested with trypsin and analyzed by LC-UVPD-MS/MS in the negative nanoelectrospray mode to generate a, c, x, y, and z product ions, which are defined in the fragmentation key in the upper left corner of the spectrum. SO₃ is retained on all product ions allowing the sulfate modification to be localized to Y₄₁. Neutral losses of SO₃ from the precursor ion and charge reduced radical precursor ion are denoted as (M-2H—SO₃)²⁻ and (M-2H—SO₃)^(1−●), respectively in the spectrum. The ion labeled “m7” refers to the neutral loss of the sulfo-tyrosine side chain without additional fragmentation of the peptide backbone. Examination of extracted ion chromatograms of the sulfated and non-sulfated peptides suggest that the sulfated peptide is 100× lower in abundance than the non-sulfated peptide. (see FIG. 23). (FIGS. 12C-D) RaxX-His proteins purified from PXO99(praxX-His) and PXO99ΔraxST(praxX-His) were analyzed by selected reaction monitoring-mass spectrometry (SRM-MS) (see FIG. 22). Total peak areas (arbitrary units) were quantitated for sulfated (FIG. 12C) and nonsulfated (FIG. 12D) tryptic RaxX peptides covering Y41.

FIGS. 13A-13D. Sulfated RaxX triggers XA21-mediated defense responses. (FIG. 13A) The 60 amino acid RaxX sequence is shown. RaxX derivative peptides of varying lengths were tested for their ability to activate XA21-dependent signaling. Sulfated peptides (RaxX_60, RaxX_39, RaxX_24 and RaxX_21) trigger XA21-mediated defense responses, whereas nonsulfated peptides (RaxX_18) do not. “EC50 value on Ubi-XA21” refers to the EC50 values determined by monitoring total ROS production over 3 hours following application of the RaxX protein and peptide derivatives (see FIGS. 34A-34E) at different concentrations on Kitaake rice expressing XA21 under the control of the ubiquitin promoter (Ubi::XA21) (n=6). (FIG. 13B) ROS production in leaves of Kitaake and Ubi::XA21 rice plants treated with H₂O (mock), RaxX21-Y or RaxX21-sY (250 nM) (n=3). RLU stands for relative light units. (FIG. 13C) Ethylene production in leaves of Kitaake and Ubi::XA21 rice plants after 4 hours of treatment with H₂O (mock), RaxX21-Y or RaxX21-sY (1 μM). The ‘*’ indicates statistically significant difference from mock treatment using Dunnett's test (α=0.01, n=3). (FIG. 13D) Temporal changes in defense marker gene (Os04g10100, PR10b, and Os12g36830) expression in leaves of Kitaake and Ubi::XA21 rice plants treated with H₂O (mock), RaxX21-Y or RaxX21-sY (500 nM, n=3). All data points depict means±SE. These experiments were repeated at least 3 times with similar results.

FIGS. 14A-14E. Comparative genomics and mutational analyses identify key amino acids required for RaxX activity. (FIG. 14A) Alignment of amino acid sequences of RaxX from PXO99 and IXO685. The region corresponding to RaxX21 is boxed. (FIGS. 14B-C) RaxX from the field strain IXO685 does not trigger XA21-mediated immune response. (FIG. 14B) TP309 (open bars) and XA21-TP309 (black bars) were inoculated with the indicated Xoo strains, and lesion lengths were measured 14 days later as described in FIG. 11B. Bars indicate the mean lesion length±SE (n≥14). (FIG. 14C) ROS production in leaves of Kitaake (open bars) and Ubi::XA21 (black bars) rice plants treated with the indicated peptides (250 nM) or mock treatment. Bars depict mean relative light production (RLU) over 3 h±SE (n=6). (FIGS. 14D-E) RaxX point mutation analysis reveals that P44 and P48 are required for activation of XA21-mediated immune response. (FIG. 14D) TP309 (open bars) and XA21-TP309 (black bars) were inoculated with the indicated Xoo strains, and lesions were measured 14 days later as described in FIG. 11B. Bars indicate the mean lesion length±SE (n≥14). (FIG. 14E) ROS production in leaves of Kitaake (open bars) and Ubi::XA21 (black bars) rice plants treated with the indicated peptides (250 nM) or mock treatment. Bars depict average relative light production ±SE (n=6). For FIGS. 14B-D, the ‘*’ indicates statistically significant difference from mock treatments using Dunnett's test (α=0.01). These experiments were repeated at least 3 times with similar results.

FIGS. 15A and 15B. Sequence alignment of 15 raxX alleles. raxX sequences were identified and aligned for the following Xanthomonas strains: X. translucens DAR61454, X. vesicatoria pv. vasculorum NCPPB206, X. campestris pv. musacearum NCPPB4394, X. campestris pv. vasculorum NCPPB792, X. sp. M97, X. axonopodis pv. manihotis UA303, X. axonopodis pv. alfalfae CFBP3836, X. euvesicatoria 85-10, X. perforans 91-118, X. axonopodis pv. citrumelo F1, X. oryzae pv. oryzae PXO99, X. oryzae pv. oryzae ATCC35933, X. oryzae pv. oryzae IXO685, X. oryzae pv. oryzicola BLS256, and X. oryzae pv. oryzicola MAI10. The alignment was created using Geneious software (Kearse M, et al. (2012) Bioinformatics 28(12):1647-1649). The raxX sequence from IXO685 was determined by Sanger sequencing. All other sequences were downloaded from NCBI, which was last accessed Dec. 14, 2014.

FIG. 16. raxX is required for activation of XA21-mediated immunity in the Ubi::XA21 Kitaake (Ubi::XA21) genetic background. Kitaake (open bars) or Ubi::XA21 (black bars) were inoculated by clipping with scissors dipped in Xoo suspensions (10⁸ CFU/mL). Bars indicate the mean lesion length±SE 14 days after inoculation (n≥4). The ‘*’ indicates statistically significant difference from PXO99 using Dunnett's test (α=0.01). There were no statistical differences in lesion length on Kitaake inoculated with the four strains.

FIG. 17. Alignment of the PAPS binding motif of RaxST from PXO99, TPST1 from human, and TPST2 from human. The conserved PAPS binding motif is boxed, and the conserved arginine in the PAPS biding motif is noted with an asterisk.

FIGS. 18A to 18B. RaxX Y41 is required for activation of XA21-mediated immunity. In planta bacterial growth analysis was carried out as described (Bahar O, et al. (2014) PeerJ 2:e242). Bacteria were extracted from leaves 0, 3, 6, 10, and 14 days after inoculation and plated on selective media. Colonies were counted after 2 days incubation at 28° C. Data points indicate the mean±SE (n=4). For the final time point, the ‘*’ indicates a statistically significant difference from PXO99 using Dunnett's test (α=0.01). This experiment was performed twice with similar results.

FIG. 19. RaxX Y41F does not trigger the XA21-mediated immune response. Total ROS production over 3 h in response to chemically synthesized sulfated RaxX peptide variants. Leaf strips of Kitaake and Ubi::XA21 were treated with the indicated peptide (250 nM) for 3 h. Bars depict mean relative light production (RLU)±SE (n=6). This experiment was repeated four times with similar results.

FIG. 20. Purification of the full-length RaxST protein carrying an N-terminal His-tag. RaxST was expressed with an N-terminal His-tag (His-RaxST) in E. coli BL21(DE3) and purified using Ni-NTA agarose beads. The purified His-RaxST was resolved by SDS-PAGE for both Coomassie-blue staining (CB) and western blot analysis with an anti-His antibody (WB). The purified His-RaxST was detected at the predicted size of 32 kDa.

FIG. 21. Expanded MS1 spectrum of HVGGGDsYPPPGANPK from trypsin digestion of in vitro sulfated RaxX39. Negative mode MS1 spectrum expanded between m/z 769 and 772 showing an accurate mass measurement for doubly deprotonated HVGGGDsYPPPGANPK (2-) (SEQ ID NO:4)acquired using an Orbitrap Elite mass spectrometer operated at an FT resolution of 120,000 at m/z 400.

FIG. 22. Extracted ion chromatograms (XIC) showing the difference in ion abundances for the sulfated and nonsulfated peptide HVGGGDYPPPGANPK (2-charge state; SEQ ID NO:4) After incubation with E. coli-expressed and purified His-RaxST, RaxX39 was digested with trypsin and analyzed by LC-UVPD-MS/MS (FIG. 12B). XICs of the sulfated (lower panel) and non-sulfated (upper panel) peptides suggest that the sulfated peptide is ˜100× lower in abundance than the non-sulfated peptide. Nonsulfated peptide and sulfated peptide are marked by arrows.

FIG. 23. Positive UVPD mass spectrum of HVGGGDsYPPPGANPK (SEQ ID NO:4) from trypsin digestion of in vitro sulfated RaxX39. 30 MS/MS scans were averaged during peptide elution between 14.23-14.53 min. Two pulses at 2 mJ was used for fragmentation. Neutral loss of SO₃ from the precursor ion is denoted as (—SO₃)²⁺. All product ions containing the tyrosine residue undergo the neutral loss of SO₃; these ions are denoted in red font.

FIG. 24. RaxX-His proteins were purified from PXO99(praxX-His) and PXO99ΔraxST(praxX-His). PXO99(praxX-His) and PXO99ΔraxST(praxX-His) were grown in XVM2 media for 40 h at 28° C. RaxX-His proteins were purified from Xoo cell lysates. The purified RaxX-His proteins were resolved by SDS-PAGE and subjected to western blot analysis with an anti-His antibody.

FIGS. 25A-25D. Expression and sulfation of RaxX60-Y and RaxX60-sY. (FIG. 25A) Purity assessment of 1 μg of RaxX60-Y and RaxX60-sY by coomassie colloidal blue stain (SimplyBlue SafeStain). RaxX60-Y and RaxX60-sY proteins purified from E. coli were analyzed by selected reaction monitoring-mass spectrometry (SRM-MS). Total peak areas (arbitrary units) were quantitated for an unmodified internal control peptide [K.GRPEPLDQR.L] (FIG. 25B) as well as nonsulfated (FIG. 25C) and sulfated (FIG. 25D) tryptic RaxX peptides [K.HVGGGDYPPPGANPK.H] covering Y41 (SEQ ID NO:4).

FIGS. 26A and 26B. SRM-MS analysis of the tryptic RaxX peptide HVGGGDYPPPGANPK from PXO99(praxX-His) and PXO99 ΔraxST(praxX-His). RaxX-His was isolated from PXO99(praxX-His) and PXO99ΔraxST(praxX-His), digested with trypsin and analyzed by SRM-MS as described in the materials and methods. (FIG. 26A) LC-SRM-MS chromatograms of the doubly and triply charged nonsulfated peptide HVGGGDYPPPGANPK (SEQ ID NO:4). (FIG. 26B) LC-SRM-MS chromatograms of the doubly and triply charged sulfated peptide HVGGGDsYPPPGANPK(SEQ ID NO:4). The lines correspond to individual SRM transitions that were monitored for each peptide. The peptide y-series fragment ion detected is indicated in the legend of each panel. This experiment was repeated twice with similar results.

FIGS. 27A and 27B. Heterologously expressed, full length RaxX60-sY activates XA21-mediated defense gene expression. Defense gene (PR10b and Os04g10100) expression changes in leaves of Kitaake (FIG. 27A) and Ubi::XA21 (FIG. 27B) rice plants treated with H₂O (mock), RaxX60-Y or RaxX60-sY (500 nM) for 24 h. Gene expression levels were measured by qRT-PCR and normalized to actin. Bars depict mean expression level±SD (n=2). This experiment was repeated at least 6 times with similar results.

FIGS. 28A and 28B. Chemically synthesized RaxX39-sY activates XA21-mediated defense gene expression. Defense gene (PR10b (FIG. 28A) and Os06g37224 (FIG. 28B)) expression changes in leaves of Kitaake and Ubi::XA21 rice plants treated with H₂O (mock), RaxX39-Y or RaxX39-sY (500 nM) for 24 h. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Bars depict mean expression level±SD (n=2). This experiment was repeated at least 9 times with similar results.

FIGS. 29A-29D. Digestion of RaxX39-sY by the four site-specific proteases GluC, trypsin, ArgC and ApsN. (FIG. 29A) Tris-tricine gel of digestion products (4 μg) of RaxX39-sY treated with the indicated protease and stained with coommassie colloidal blue stain (SimplyBlue SafeStain). Digestion products of trypsin, ArgC and AspN were not resolved on tris-tricine gels due to small digestion product sizes (<2.6 kDa). Appropriate digestion for these three proteases was confirmed by selected reaction monitoring-mass spectrometry (SRM-MS). (FIGS. 29B-D) shows representative LC-SRM-MS chromatograms of trypsin [K.HVGGGDsYPPPGANPK.H] (SEQ ID NO:4), ArgC [R.LWKHVGGGDsYPPPGANPKHDPPPR.N], and AspN [G.DsYPPPGANPKH.D] RaxX digestion products covering the sulfated Y41 residue. The lines correspond to individual SRM transitions that were monitored for each peptide. The peptide y-series fragment ion detected is indicated in the legend at the top of the plot. This experiment was repeated twice with similar results.

FIGS. 30A-30B. A 21 amino acid sulfated peptide derived from RaxX is sufficient to activate XA21-mediated defense gene expression and the production of reactive oxygen species. (FIG. 30A) Gene (Os06g37224) expression changes in leaves of Ubi::XA21 rice plants treated with protease digestion buffer (mock), RaxX39-sY, RaxX39-Y, or digestion products thereof produced using the indicated site specific proteases. Leaf strips were treated with peptides and their digests (1 μM) for 24 h. Expression levels were measured by qRT-PCR and normalized to actin. Bars depict mean expression level±SD (n=2). This experiment was repeated 3 times with similar results. (FIG. 30B) Total ROS production over 3 h in response to chemically synthesized sulfated RaxX peptide variants. Leaf strips of Kitaake and Ubi::XA21 were treated with the indicated peptide (250 nM) for 3 h. Bars depict mean relative light production (RLU)±SE (n=6). Different letters indicate statistically significant differences using Tukey HSD test (α=0.01). This experiment was repeated at least twice with similar results.

FIGS. 31A-31B. Chemically synthesized RaxX21-sY is sufficient to activate XA21-mediated defense gene expression. Temporal defense gene expression (Os06g37224) changes in leaves of Kitaake (FIG. 31A) and Ubi::XA21 (FIG. 31B) rice plants treated with H₂O (mock), RaxX21-Y or RaxX21-sY (500 nM). Data points depict mean expression level±SE (n=3). This experiment was repeated at least 3 times with similar results.

FIG. 32. Chemically synthesized RaxX21-sY activates gene expression in Kitaake lines expressing XA21 from its native promoter (XA21:XA21). Temporal gene expression changes in leaves of five week old XA21::XA21 rice plants treated with H₂O (mock) or RaxX21-sY (500 nM). Data points depict mean expression level±SD (n=2). This experiment was repeated at least 3 times with similar results.

FIGS. 33A and 33B. Chemically synthesized RaxX21-sY activates the production of ROS in Kitaake lines expressing XA21 from its native promoter (XA21:XA21). ROS production in five-week (FIG. 33A) and six-week (FIG. 33B) old leaves of Kitaake, Ubi::XA21 and XA21::XA21 rice plants treated with H₂O (mock), RaxX21-Y or RaxX21-sY (250 nM). Data points depict mean ROS production±SE (n=6). This experiment was repeated 3 times with similar results.

FIGS. 34A-34E. ROS production dose response curves in Ubi::XA21 leaves treated with chemically synthesized RaxX sulfated peptides. Individual data points depict single measurements of total ROS production over 3 h in leaves of five-week old Ubi::XA21 rice plants treated with the indicated peptides (10 pmol to 3 μM). The fitted curve depicts the best-fitted model to the non-linear logistic 4p formula using the JMP software package. The vertical line indicates the inferred EC50 value. These experiments were repeated at least twice with similar results. RaxX60-sY (FIG. 34A), RaxX39-sY (FIG. 34B), RaxX24-sY (FIG. 34C), RaxX21-sY (FIG. 34D), and RaxX18-sY (FIG. 34E)

FIGS. 35A and 35B. The tyrosine sulfated peptide axY(S)22, derived from Ax21, does not trigger XA21-mediated immune responses in rice. Defense gene induction (Os06g37224) (FIG. 35A) and ROS production (FIG. 35B) in five-week old leaves of Kitaake and Ubi::XA21 rice plants treated with H₂O (mock), axY(S)22 (1 μM), RaxX21-Y or RaxX21-sY (250 nM). Data points depict mean expression level±SD (n=2) or mean ROS production±SE (n=6). These experiments were repeated at least 3 times with similar results.

FIG. 36. Expression and purification of different RaxX alleles when heterogously expressed in E. coli. RaxX60-sY and the indicated variants (2 μg) were expressed and purified as described in the material and methods. The purified proteins were analyzed using a Tris-tricine gel (16.5%) and stained with SimplyBlue SafeStain.

FIG. 37. Complementation of Xoo isolates IXO651, IXO685, and IXO1221 with praxX confers upon these strains the ability to activate XA21-mediated immunity. TP309 (open bars) or XA21-TP309 (black bars) were inoculated by clipping with scissors dipped in Xoo suspensions (10⁸ CFU/mL). Bars indicate the mean lesion length±1 standard error (SE) 14 days after inoculation (n≥4). The ‘*’ indicates statistically significant difference using Dunnett's test (α=0.01).

FIGS. 38A and 38B. RaxX is similar to the Arabidopsis thaliana peptide signaling factors PSY1 and to rice PSY1 homologs. (FIG. 38A) Alignment of RaxX21 from PXO99 with the 18 amino acid Arabidopsis plant peptide containing sulfated tyrosine 1, PSY1(AT5g58650), and two homologs from Arabidopsis (Amano Y et al. (2007) Proc Natl Acad Sci USA 104(46):18333-18338). The numbering corresponds to the full length RaxX sequence. (FIG. 35B) Alignment of RaxX21 with 4 rice PSY1 homologs. These PSY1 rice homologs were identified by BLAST analysis using the 18 amino acid sequence of processed PSY1. Four rice genes encoding PSY1 like sequences were identified: Os01g16980, Os01g59970, Os05g40890, and Os05g40850. Os05g40850 carries four tandem repeats containing highly similar PSY1-like sequences, labeled Os05g40850-A-D. Alignments of the amino acid sequences corresponding to the processed PSY1 peptide with RaxX21 were generated using Geneious software (Kearse M, et al. (2012) Bioinformatics 28(12):1647-1649).

FIG. 39A-B. Sequence similarity of RaxX and plant PSYs.a) The sequence of RaxX21 from Xoostrain PXO99 was aligned with the 18 amino acid AtPSY1 and a PSY-like repeat of OsPSY1.Endogenous AtPSY1 has 3postranslationally modified residues (shown in red): a sulfotyrosine and two hydroxyprolines. The first hydroxyproline is further modified by chain of three L-arabinose residues(L-Ara₃).The synthetic RaxX peptides RaxX16 and RaxX13 are truncated relative to RaxX21 as depicted. b) Sequence logos depicting the amino acid composition in the conserved 13-amino acid region of RaxX and PSY proteins. The logos were generated from 34 PSY sequences and 17 non-redundant RaxX13 sequences (Table S1).

FIG. 40A-F. Sulfated RaxX21 promotes root growth in Arabidopsis and rice. Root lengths of Arabidopsis a) Col-0 or c)tpst-1 seedlings were grown on 0.5× MS vertical plates with or without 100 nM of the indicated peptides. Bars indicate the average seedling root length measured after 8 days (n≥18).b and d) 8-day old Col-0 and tpst-1 seedlings grown as in 40a and c, respectively. e) Growth rate of six day old tpst-1 seedlings following transfer to 0.5× MS plates containing 250 nMRaxX21-sY, 250 nM AtPSY1, or lacking peptide (Mock) (n≥7). Growth was monitored by continual imaging over 20 h.f) Root lengths of 6-day old rice seedlings (Tapei 309) grown on 0.5× MS with or without 100 nM of the indicated peptides (n≥37). Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05).

FIGS. 41A and 41B. The Arabidopsisgene Atlg72300 is not required for RaxX- and PSY-induced root growth. a) Arabidopsis Atlg72300 or b) AtPSKR1/AtPSKR2/Atlg72300 triple receptor mutant seeds were grown on 0.5× MS plates with or without 100 nM of the indicated peptides. Root lengths were measured 8 days after placing seeds on plates. Error bars indicate standard error (n≥10). Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05). The experiment was performed at least three times with similar results.

FIGS. 42A to 4D. Differential activities of PSY and RaxX peptides ingrowth promotion and activation of XA21-mediated immunity. ROS production in leaves of a) XA21 rice (XA21-Kitaake) and b) wild type rice (Kitaake) treated with H₂O (Mock) or 500 nM of the indicated peptide. Bars represent average ROS production over 90 min following addition of peptide (n=6). RLU stands for relative light units. c) TP309seeds were grown on 0.5× MS media for with or without 100 nM of the indicated peptides. Root lengths were measured 5 days after placing seeds on plates (n≥25). d) Arabidopsistpst-1 seeds were grown on 0.5× MS vertical plates with or without 100 nM of the indicated peptides. Root lengths were measured 8 days after placing seeds on plates (n≥16). Error bars indicate standard error. Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05). Experiments were performed at least two times with similar results.

FIG. 43. RaxX peptides derived from RaxX encoded by Xoc, Xe, and Xcm promote root growth in Arabidopsis seedlings. tpst-1 seedlings were grown on 0.5× MS vertical plates with or without 100 nM of the indicated peptides. Bars indicate the average seedling root length measured after 8 days (n≥18). Error bars indicate error. Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05). Experiments were performed at least two times with similar results.

FIG. 44. Proposed model of RaxX production and activation of PSY and XA21 signaling. PSY is produced and detected by plant cells to regulate growth. RaxX is produced in Xoo, sulfated by RaxSt, and secreted by a type I secretion system composed of RaxA, RaxB, and RaxC. Secreted sulfated RaxX binds to the endogenous PSY receptor(s) and activates PSY signaling. The wild rice O. longistaminata subsequently evolved the immune receptor XA21 which selectively recognizes RaxX, but not endogenous PSY peptides.

FIG. 45A and FIG. 45B. Putative PSY-like proteins from Arabidopsis (At), rice (Os), banana (Ma), tomato (Sl), and wheat (Ta). PSY-like proteins were identified as described in the Material and Methods. The sequences are aligned based on the highly conserved 13-amino acid region.The conserved tyrosine, which is sulfated in AtPSY1, is highlighted in yellow. OsPSY1 and AtPSY8 have multiple repeats of the PSY-like motif within the full-length protein labeled with an appended letter. AtPSY8 has one pseudo-repeat lacking the tyrosine (AtPSY8x). The individual repeats are noted with an appended lowercase letter. For comparison, the RaxX sequences from X. oryzaepv. oryzae strain PXO99 and X. translucens strain 4699 are also included in the alignment. The residues of the conserved 13-amino acid region are numbered at the top of the FIG., with positions 5, 7, and 9 in red. These three residues are important for recognition of RaxX by XA21.

FIG. 46. Comparison of the RaxX sequences from diverse bacterial strains. RaxX sequences from X. oryzae (blue), X. translucens (green) and other Xanthomonas strains (orange). Amino acids are colored according to conservation among Xanthomonas strains and among all PSY-like proteins. The invariant tyrosine is highlighted in yellow. The residues of the conserved 13-amino acid PSY-like motif are numbered at the top of the FIG., with amino acids in positions 5, 7, and 9 in red. These three residues are important for recognition of RaxX by XA21.

FIG. 47. Dose dependent activity of RaxX21-Y, RaxX21-sY, AtPSY1, and PSK on root growth of Arabidopsis tpst-1 seedlings. Arabidopsis tpst-1 seeds were grown on 0.5× MS plates with or without the indicated peptides as described in the materials and methods. Root lengths were measured 8 days after plating seeds. Error bars indicate standard error (n≥16). Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05).

FIG. 48. RaxX, AtPSY1, and PSK do not have additive effects on root growth. tpst-1 seedlings were grown on 0.5× MS vertical plates with or without 100 nM of each of the indicated peptides. Bars indicate the average seedling root length measured 8 days after plating seeds (n≥18). Error bars indicate error. Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05). Experiments were performed at least two times with similar results.

FIG. 49. Sulfated RaxX21 promotes root growth in Kitaake rice. a) Root lengths of 4-day old rice seedlings (Kitaake cultivar) grown on 0.5× MS with or without 100 nM of the indicated peptides (n≥35). b) Rice seedling grown as in a) were photographed. Error bars indicate error. Statistical analysis was performed using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters represent significant differences within each plant genotype (p≤0.05). Experiments were performed at least two times with similar results.

FIG. 50. Validation of the Atlg72300 mutants.a) The Atlg72300 mutants were confirmed by PCR using the SIGnAL T-DNA Primer Design protocol (http://signal.salk.edu/tdnaprimers.2.html).Wild typeAtlg72300or the SAL_072802.48.70.x polymorphism (T-DNA insertion in At1 g72300) were detected by PCR with the following primers: LBb1.3, 5′-ATTTTGCCGATTTCGGAAC-3′; LP, 5′-GAGAACCTTTAGCTGCCCAAC-3′; RP, 5′-ACAATGATTTCAGCGGTGATC-3.′b)Atlg72300 transcript levels were assessed by qRT-PCR. 8-day old Col-0 or Atlg72300 (SALK_072802 C). RNA was treated with DNAseI, and cDNA was synthesized using MultiScribe (Applied Biosystems).qPCR reactions were performed with iTaq (BioRad) with annealing and amplification at 55° C. for 5 s and denaturation at 95° C. for 10 srun for 40 cycles. The following primers were used for qPCR: Atlg72300:5′-GAGAACCTTTAGCTGCCCAAC-3′, 5′-GCAATACAATCAGCTTTCCAGCC-3′, Actin: 5′-GGTAACATTGTGCTCAGTGGTGG-3′, 5′-GGTGCAACGACCTTAATCTTCAT-3′) Transcript levels are normalized to actin, and relative to wild-type.

FIG. 51. Sequence similarity of RaxX from Xoo and Xoc with selected rice PSYs. 13 amino acid RaxX sequences from Xoo (strain PXO99) and Xoc (strain BSL256) were aligned with the corresponding 13 amino acid fragments of OsPSY1a and OsPSY2, respectively. RaxX residues 44, 46, and 48 (marked in red boxes) are important for activation of XA21-mediated immunity.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Provided herein is a newly characterized peptide, RaxX, that activates pathogen responses and root growth in plants.

Gram negative bacterial infections pose an increasing problem due in part to appearance of antibiotic resistant strains. These bacteria are particularly persistent, at least in part because they can grow within biofilms that protect them from adverse environmental factors.

Xanthomonas oryzae pv. oryzae (Xoo) is both a serious disease of rice, an important US crop, and is a predictive model organism for studies of other Xanthomonas species, which infect virtually all plant species. Sequence analysis of the raxX-raxSTAB genomic region reveals that orthologs are present in the agronomically important pathogens X. oryzae pv. oryzicola (Xoc) (98% identity), X. campestris pv. vesicatoria (Xcv) (88% identity), X. axonopodis pv. citrumelo (Xac) (88% identity) and X. campestris pv. musacearum (Xcm) (>85% identity). The present results are thus relevant for essentially all agronomically important plant hosts, and of fundamental biological relevance to agriculture.

The present results show isolation and characterization of RaxX (Required for Activation of XA21-mediated immunity), a small protein conserved in many species of the Gram-negative bacterial genus Xanthomonas and in species of related genera. RaxX-mediated biological processes are mediated by a Type I secretion system, a tyrosine sulfotransferase, and a response regulator (e.g., RaxR). In Xanthomonas, RaxA, RaxB, CvaA, and CvaB are predicted to process and secrete RaxX.

Small molecules play a key role in bacterial infection of plants and animals. The present results demonstrate for the first time the small protein RaxX, its tyrosine sulfation by RaxST, and processing and secretion by RaxAB and CvaAB in Gram-negative bacteria. Sulfated RaxX is shown to trigger robust resistance in plants expressing XA21. Thus, the characterization of RaxX, RaxST, and the other Rax and Cva genes radically alters the conceptual framework underlying the understanding of Gram-negative bacterial infection.

The present findings have important implications for development of novel approaches to disrupt Gram-negative bacterial infection. For example, RaxX (potentially in combination with other Rax and Cva genes involved in XA21-mediated immunity in plants) can be used to develop antagonists to disrupt small protein-mediated processes in Gram-negative bacterial infection.

II. Definitions

“Enhanced disease resistance” refers to an increase in the ability of a plant to prevent pathogen infection or pathogen-induced symptoms. Enhanced resistance can be increased resistance relative to a particular pathogen species or genus or can be increased resistance to all pathogens (e.g., systemic acquired resistance). The terms “pathogen response,” “disease resistance,” “immune response,” and like terms are used interchangeably unless otherwise indicated. While plants do not have an adaptive immune system per se, one of skill in the art will understand immunity terms as commonly used with relation to plants. Disease resistance can take the form of a hypersensitive response, in which cells of the plant that detect pathogen undergo programmed cell death (see, e.g., Watanabe and Lam (2006) Multigenic and Induced Systemic Resistance in Plants p. 83). Disease resistance can also be manifested by release of antimicrobial chemicals and hormones, e.g., reactive oxygen species (H2O2, peroxynitrite, phytoalexins), defensins, thionins, chitinase, peroxidase, etc. Disease resistance can also be detected by detecting pathogen-induced or defense gene expression, e.g., as shown in FIGS. 6 and 7A to 7C.

The term “exogenous application” taken in its broadest context includes contacting or administering cells, tissues, organs or organisms with a suitable compound or element. The compound may be applied to a plant in a suitable form for uptake (such as through application to the soil for uptake via the roots, or by applying directly to the leaves, for example by spraying).

The term “sulfated tyrosine” is used to include tyrosine-O-sulfate residues comprising a sulfate group covalently bound via the hydroxyl group of the tyrosine side chain. Sulfate may be added to a tyrosine by post-translational modification of a peptide or protein (e.g., by RaxST), by chemical synthesis (e.g., incorporating a sulfonated tyrosine into a synthetically produced polypeptide), or by chemical alteration. As used herein, “Y” indicates a tyrosine residue, while “Y*” indicates a sulfated tyrosine.

“Pathogens” include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, Calif. (1988)).

The term “homolog” refers to both interspecies orthologs and intraspecies paralogs. Unless indicated otherwise, a homolog will share at least some activity (e.g., at least 25, 30, 40, 50, 60, 75, 80, 90% or higher level of activity) with the named gene or gene product (e.g., polypeptide), and thus be a “functional” homolog. Orthologs are genes in different species that typically retain the same function and evolved from a common ancestral gene prior to speciation. Paralogs are similar or related genes within a species genome. Paralogs typically arise from duplication, and often have overlapping functions.

The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The term includes constitutive, inducible, and tissue specific promoters, any of which can be used as described herein. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A plant promoter can be, but does not have to be, a nucleic acid sequence originally isolated from a plant.

The term “plant” includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a different strain or species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).

“Recombinant” refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.

The term “nucleic acid” or “polynucleotide” as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156).

The phrase “host cell” refers to a cell from any organism. Preferred host cells are derived from plants, bacteria, yeast, fungi, insects or other animals. Methods for introducing polynucleotide sequences into various types of host cells are well known in the art.

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell (e.g., a plant cell), results in transcription and/or translation of a RNA or polypeptide, respectively.

Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term “complementary to” is used herein to mean that the sequence is complementary to all or a portion of a reference polynucleotide sequence.

One example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity. Alternatively, percent identity can be any integer from 25% to 100%, e.g., at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that the percent identity values above can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 40%., e.g., any integer from 40% to 100%. Exemplary embodiments include at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, polypeptides that are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Provided herein are polynucleotides encoding a polypeptide comprising an amino acid sequence substantially identical to raxX (e.g., the polypeptide sequence of SEQ ID NO:1, allelic variants, and homologs thereof). The present invention also provides for a polypeptide comprising an amino acid sequence substantially identical to raxX.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

III. Polypeptides Involved in Pathogen Resistance and Root Growth and Uses Thereof

Provided herein are small peptide molecules that are conserved across species that can be used as targets for inhibiting Gram negative and nosocomial bacterial infection. Such polypeptides include raxX and raxX-like polypeptides.

RaxX (and variants and homologs thereof) can be used to activate immunity in plants expressing XA21, and to develop antagonists that disrupt raxX function in bacteria. Such antagonists can then be used as antibiotics to combat infection in plants, animals, etc.

The polypeptides described herein have a number of uses. Notably, contacting a plant with a polypeptide as described herein can induce or enhance disease resistance in a plant. Moreover, the inventors have found that contacting a plant with a polypeptide as described herein enhances root growth. The contacting can occur by applying exogenous (not expressed in the plant) polypeptide to the plant, or by expressing the polypeptide in the plant.

In some embodiments, the plants express XA21 or a functional equivalent, i.e., a disease resistance gene product that enables recognition of a polypeptide as described herein, and subsequently induces disease resistance in the plant. The XA21 gene was first isolated from rice. See, e.g., Song, W. et al., Science 270:1804 (1995); and U.S. Pat. No. 5,977,434. The plant can express XA21 endogenously (e.g., not by recombinant expression) or the plant can be transgenic or otherwise recombinantly manipulated to express XA21 (e.g., heterologous XA21 from a different plant strain or species). Thus, while XA21 was originally identified in rice, XA21 can be expressed in plants other than rice. Plants other than rice have XA21 activity, and can be identified, e.g., by enhanced disease resistance in response to contact with a polypeptide as described herein.

Provided herein are methods of identifying a plant having XA21 activity by contacting a non-rice plant with a polypeptide as described herein (either recombinantly produced, purified, or expressed from a bacterial or fungal pathogen) and identifying a contacted plant that has enhanced disease resistance or other manifestation of disease resistance (e.g., a hypersensitive response) as a result the presence of the polypeptide in the contacting step.

Small proteins such as raxX are an attractive therapeutic target because of the important role that they play in the global regulation of multiple bacterial factors and in the virulence of an organism at multiple different infection stages. Quorum sensing (QS) is often mediated by small proteins, so disruption of the small protein interactions can prevent pathogenic bacteria from initiating the gene expression cascade required for successful establishment in the host. Bacterial mutants impaired in QS are attenuated in virulence (Jayaraman et al. (2008) Annu Rev Biomed Eng 10:145).

A common problem associated with antibiotic control of infection is that the chemical reduces the growth of the bacteria. This strategy increases selection pressure on the bacteria to evolve mechanisms to resist the chemical. In contrast, it is more difficult for the bacteria to evolve or change its QS system because in order for the bacteria to no longer bind to the antagonist it would need to mutate the binding domain of the QS receptor. In this case, however, the receptor would no longer be able to bind the endogenous QS factor and would therefore be crippled in virulence. Even if a bacterium successfully mutated the receptor to no longer bind the antagonist but was able to retain binding with the endogenous QS molecule, other bacteria in the population would still be susceptible to the antagonist. Consequently, a bacterium with resistance to a chemical antagonist has no growth advantage compared with neighboring bacteria and is not able to outgrow other bacteria in subsequent generations. Anti-QS strategies thus offer the attractive possibility that any mutations to resist the chemical would also reduce bacterial fitness, giving these chemicals a long functional lifetime.

Provided herein are methods of screening for compounds that antagonize RaxST or RaxX density dependent expression in Gram negative bacteria (e.g. Xanthomonas). Test compounds can be obtained from libraries of characterized natural compounds and synthetic derivatives (e.g. NCI, Spectrum, and Enzo libraries). In some embodiments, highly antagonistic compounds from the first screen (e.g., at least 2 or 3 standard deviations from negative control values) are further screened for inhibition of raxX-mediated and/or raxST-mediated virulence of pathogens in plant cells.

This screen identifies molecules that have the potential to affect several different stages of the raxX-mediated signaling pathway (e.g. raxX sulfation, secretion, uptake, transmission of signal from the sensor to kinase domain, transfer of signal to the response regulators, etc), not just small molecules that disrupt protein-protein interactions. Established assays can then be used to determine the effect of candidate compounds on RaxX secretion, processing and sulfation (e.g., Han and Lee (2012) Nature Comm). Similar screening methods can be carried out for raxX variants and raxX species homologs to investigate and screen for inhibitors in other pathogen-host systems, including infections of Gram negative bacteria (and antibiotic resistant strains) in humans.

Bacteria, including bacterial causal agents of animal and human disease, rely on various processes for virulence. Administration of a therapeutically effective amount of a polypeptide as described herein (e.g., raxX and raxX-like peptides), dominant-negative variants, and/or inhibitors obtained from the above screening methods, can disrupt virulence functions of bacterial animal or human pathogens in animals (e.g., bovines, poultry animals, pigs, sheep, dogs, cats, horses, rats, mice, etc.) or humans, thereby treating or ameliorating disease caused by such bacterial agents. Variants and inhibitors of the presently disclosed peptides that block endogenous bacterial processes required for virulence are of particular interest. Such treatment is expected to be effective against any bacterial animal or human pathogen that uses a polypeptide substantially identical to a RaxX polypeptide of the invention as a QS agent. Exemplary bacterial species include, but are not limited to, Staphylococcus aureus, Bordetella pertussis, Stenotrophomonas maltophilia, Lactobacillus plantarum and Lactobacillus sake.

Administration of a polypeptide or inhibitory molecule as described herein can be by any of the routes normally used for introducing pharmaceuticals. The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, intrathecally or into the eye (e.g., by eye drop or injection). The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.

The dose administered to a patient should be sufficient to induce a beneficial response in the subject over time, i.e., reduce pathogen infection and effects. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, and on a possible combination with other drug. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject. Administration can be accomplished via single or divided doses.

IV. Agricultural Applications

Provided herein are agricultural formulations formulated for contacting to plants, wherein the formulation comprises a polypeptide (e.g., a sulfated polypeptide as described herein). The formulations can be suitable for treating plants or plant propagation material, such as seeds, in accordance with the present invention, e.g., in a carrier. Suitable additives include buffering agents, wetting agents, coating agents, polysaccharides, and abrading agents. Exemplary carriers include water, aqueous solutions, slurries, solids and dry powders (e.g., peat, wheat, bran, vermiculite, clay, pasteurized soil, many forms of calcium carbonate, dolomite, various grades of gypsum, bentonite and other clay minerals, rock phosphates and other phosphorous compounds, titanium dioxide, humus, talc, alginate and activated charcoal). Any agriculturally suitable carrier known to one skilled in the art would be acceptable and is contemplated for use in the present invention. The formulations can also include at least one surfactant, herbicide, fungicide, pesticide, or fertilizer.

Treatment can be performed using a variety of known methods, e.g., by spraying, atomizing, dusting or scattering the compositions over the propagation material or brushing or pouring or otherwise contacting the compositions over the plant or, in the event of seed, by coating, encapsulating, or otherwise treating the seed. The formulations described herein can also or alternatively be introduced into the soil or other media into which the seed is to be planted. In some embodiments, a solid or liquid carrier is used. In some embodiments, peat is suspended in water as a carrier of the desired polypeptide, and this mixture is sprayed into the soil or planting media and/or over the seed as it is planted.

V. Recombinant Techniques and Compositions

To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A polynucleotide sequence encoding a desired polypeptide (e.g., a polypeptide as described herein, e.g., including but not limited to XA21, raxB, raxST, raxX, and functional fragments, allelic variants, and homologs thereof), will be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the polynucleotid in the intended tissues of the transformed plant.

For example, a plant promoter can be employed which will direct expression of the polynucleotide in all tissues of a transgenic plant, in a specific tissue (tissue-specific promoters), organ (organ-specific promoters), or under certain conditions (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, flowers, pistils, or anthers. Suitable promoters include those from genes encoding storage proteins or the lipid body membrane protein, oleosin.

A polyadenylation region at the 3′-end of the coding region can also be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions) from coding sequences described herein will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.

Constitutive Promoters

A promoter, or an active fragment thereof, can be employed which will direct expression of a nucleic acid encoding a fusion protein of the invention, in all transformed cells or tissues, e.g., as those of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include those from viruses which infect plants, such as the cauliflower mosaic virus (CaMV) 35S transcription initiation region (see, e.g., Dagless Arch. Virol. 142:183-191 (1997)); the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens (see, e.g., Mengiste supra (1997); O'Grady Plant Mol. Biol. 29:99-108) (1995)); the promoter of the tobacco mosaic virus; the promoter of Figwort mosaic virus (see, e.g., Maiti Transgenic Res. 6:143-156) (1997)); actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang Plant Mol. Biol. 33:125-139 (1997)); alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar Plant Mol. Biol. 31:897-904 (1996)); ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)), other transcription initiation regions from various plant genes known to those of skill. See also Holtorf (1995) “Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana,” Plant Mol. Biol. 29:637-646.

Inducible Promoters

Alternatively, a plant promoter may direct expression of the nucleic acids under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Examples of developmental conditions that may affect transcription by inducible promoters include senescence and embryogenesis. Such promoters are referred to herein as “inducible” promoters.

Exemplary inducible promoters include those promoters that are specifically induced upon infection by a virulent pathogen. Useful promoters include PR10b, Os06g13390, Os04g10010, Os02g36190.1, Os04g09920.1, Os06g28050, Os06g37224.1, Os10g28299, Os11g31550.1, Os12g36830.1, those discussed in PCT application WO 99/43824, and promoters from:

-   a. pathogen response genes (e.g., Lee et al. (2011) PNAS 108:18548); -   b. lipoxygenases (e.g., Peng et al, J. Biol. Chem. 269:3755-3761     (1994)), -   c. peroxidases (e.g., Chittoor et al. Molec. Plant-Microbe Interact.     10:861-871 (1997)), -   d. hydroxymethylglutaryl-CoA reductase, -   e. phenylalanine ammonia lyase, -   f. glutathione-S-transferase, -   g. chitinases (e.g., au et al. Mol. Gen. Genet. 226:289-296 (1991)), -   h. genes involved in the plant respiratory burst (e.g., Groom et al.     Plant J. 10:515-522 (1996)); and -   i. pathogenesis-related (PR) protein promoters.

Other examples of developmental conditions include cell aging, and embryogenesis. For example, the invention incorporates the senescence inducible promoter of Arabidopsis, SAG 12, (Gan and Amasino, Science, 270:1986-1988 (1995)) and the embryogenesis related promoters of LEC1 (Lotan et al., Cell, 93:1195-205 (1998)), LEC2 (Stone et al., Proc. Natl. Acad. of Sci., 98:11806-11811 (2001)), FUS3 (Luerssen, Plant J. 15:755-764 (1998)), AtSERK1 (Hecht et al. Plant Physiol 127:803-816 (2001)), AGL15 (Heck et al. Plant Cell 7:1271-1282 (1995)), and BBM (BABYBOOM). Other inducible promoters include, e.g., the drought-inducible promoter of maize (Busk supra (1997)) and the cold, drought, and high salt inducible promoter from potato (Kirch Plant Mol. Biol. 33:897-909 (1997)).

Plant promoters which are inducible upon exposure to plant hormones, such as auxins or cytokinins, can be used to express the nucleic acids of the invention. For example, the invention can use the auxin-response elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu Plant Physiol. 115:397-407 (1997)); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen Plant J. 10:955-966 (1996)); the auxin-inducible parC promoter from tobacco (Sakai 37:906-913 (1996)); a plant biotin response element (Streit Mol. Plant Microbe Interact. 10:933-937 (1997)); and, the promoter responsive to the stress hormone abscisic acid (Sheen Science 274:1900-1902 (1996)). The invention can also use the cytokinin inducible promoters of ARRS (Brandstatter and Kieber, Plant Cell, 10:1009-1019 (1998)), ARR6 (Brandstatter and Kieber, Plant Cell, 10:1009-1019 (1998)), ARR2 (Hwang and Sheen, Nature, 413:383-389 (2001)), the ethylene responsive promoter of ERF1 (Solano et al., Genes Dev. 12:3703-3714 (1998)), and the β-estradiol inducible promoter of XVE (Zuo et al., Plant J, 24:265-273 (2000)).

Plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics, are also used to express the nucleic acids of the invention. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder Plant Cell Physiol. 38:568-577 (1997)) as well as the promoter of the glucocorticoid receptor protein fusion inducible by dexamethasone application (Aoyama, Plant J., 11:605-612 (1997)); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. The coding sequence of the described nucleic acids can also be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau Plant J. 11:465-473 (1997)); or, a salicylic acid-responsive element (Stange Plant J. 11:1315-1324 (1997)).

Tissue-Specific Promoters

Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots, leaves or stems, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistils, flowers, or any embryonic tissue.

A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can also be used to express the nucleic acids used in the methods of the invention. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used, e.g., Kim Plant Mol. Biol. 26:603-615 (1994); Martin Plant J. 11:53-62 (1997). The ORF13 promoter from Agrobacterium rhizogenes which exhibits high activity in roots can also be used (Hansen Mol. Gen. Genet. 254:337-343 (1997)). Other useful vegetative tissue-specific promoters include: the tarin promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra Plant Mol. Biol. 28:137-144 (1995)); the curculin promoter active during taro corm development (de Castro Plant Cell 4:1549-1559 (1992)) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto Plant Cell 3:371-382 (1991)).

Leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier FEBS Lett. 415:91-95 (1997)). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka Plant J. 6:311-319 (1994), can be used. Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter, see, e.g., Shiina Plant Physiol. 115:477-483 (1997); Casal Plant Physiol. 116:1533-1538 (1998). The Arabidopsis thaliana myb-related gene promoter (Atmyb5) (Li FEBS Lett. 379:117-121 (1996)) is leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16-cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize by Busk Plant J. 11:1285-1295 (1997) can also be used.

Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the “SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in the developing shoot or root apical meristems, described by Di Laurenzio Cell 86:423-433 (1996) and Long Nature 379:66-69 (1996), can be used. Another useful promoter is that which controls the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto Plant Cell. 7:517-527 (1995)). Also useful are kn1-related genes from maize and other species which show meristem-specific expression, see, e.g., Granger Plant Mol. Biol. 31:373-378 (1996); Kerstetter Plant Cell 6:1877-1887 (1994); Hake Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51 (1995). For example, the Arabidopsis thaliana KNAT1 or KNAT2 promoters. In the shoot apex, KNAT1 transcript is localized primarily to the shoot apical meristem; the expression of KNAT1 in the shoot meristem decreases during the floral transition and is restricted to the cortex of the inflorescence stem (see, e.g., Lincoln Plant Cell 6:1859-1876 (1994)).

In some embodiments, the promoter is a root-specific promoter. This can be useful, for example, in enhancing root growth.

One of skill will recognize that a tissue-specific promoter may drive some expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein, a tissue-specific promoter is one that drives higher expression in the target tissue than in other tissues.

In some embodiments, a nucleic acid described herein can be expressed through a transposable element. This allows for constitutive, yet periodic and infrequent expression of the constitutively active polypeptide. The invention also provides for use of tissue-specific promoters derived from viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683) the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer Plant Mol. Biol. 31:1129-1139 (1996)).

VI. Transgenic Plants

Provided herein are transgenic plants. In some embodiments, the transgenic plant comprises:

a first heterologous expression cassette comprising a first promoter operably linked to a polynucleotide encoding a ligand of XA21 (e.g., comprising a sequence encoding a polypeptide substantially identical to raxX, or an active fragment thereof, e.g., with the Tyr corresponding to Tyr10 of SEQ ID NO:1 sulfonated). In some embodiments, such plants endogenously express XA21.

Optionally (e.g., when the plant does not endogenously express XA21 or have endogenous XA21-recognition activity), the plant can also comprise:

a second heterologous expression cassette comprising a second promoter operably linked to a polynucleotide encoding an XA21 polypeptide (e.g., a polypeptide substantially identical to the XA21 polypeptide described in U.S. Pat. No. 5,977,434); and/or

a third heterologous expression cassette comprising a third promoter operably linked to a polynucleotide encoding a sulfur transferase (e.g., raxST gene, or an ortholog or other active variant thereof) capable of sulfating the tyrosine Y* of the polypeptide product of the first expression cassette.

Provided herein are transgenic plants comprising the first and second expression cassettes, comprising the first and third expression cassettes, comprising the second and third expression cassettes, or all three expression cassettes.

In some embodiments, the invention provides for a transgenic plant comprising the second expression cassette contacted with an exogenous XA21 ligand (e.g., comprising a polypeptide sequence substantially identical to raxX, or an active fragment thereof, wherein the).

DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA constructs may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts. The DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment. The DNA constructs can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.

Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. (1984) Embo J. 3:2717. Electroporation techniques are described in Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824. Biolistic transformation techniques are described in Klein et al. (1987) Nature 327:70-73.

Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. (1984) Science 233:496 and Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803.

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased disease resistance compared to a control plant that was not transformed or transformed with an empty vector. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467.

The nucleic acids and encoded polypeptides of the invention can be used to confer enhanced disease resistance on essentially any plant. Thus, the invention has use over a broad range of plants, including but not limited to species of Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, eucalyptus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Setaria, Miscanthus, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.

VII. Selecting Plants with Enhanced Resistance

Plants with enhanced resistance can be selected or identified in many ways, including but not limited to the following. Plants with enhanced resistance can be identified by determining resistance of a plant to a specific plant pathogen. Possible pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, Calif.) (1988)). One of skill in the art will recognize that resistance responses of plants vary depending on many factors, including what pathogen or plant is used. Generally, enhanced resistance is measured by the reduction or elimination of disease symptoms (e.g., lesion formation) when compared to a control plant (e.g., a plant not contacted with a pathogen, a plant expressing or contacted with an immune polypeptide as described herein, and/or a plant not expressing XA21 or a protein with XA21 activity). In some cases, however, enhanced resistance can also be measured by the production of the hypersensitive response (HR) of the plant (see, e.g., Staskawicz et al. (1995) Science 268:661). Plants with enhanced resistance can produce an enhanced hypersensitive response relative to control plants.

Disease resistance can also be determined by detecting release of antimicrobial chemicals or hormones by a plant, e.g., reactive oxygen species (H2O2, peroxynitrite, phytoalexins), defensins, thionins, chitinase, peroxidase, etc.

Disease resistance can also be determined by detecting gene expression of genes that are upregulated or downregulated during pathogen response. For example, expression of the following genes is upregulated in plants fending off pathogen attack: PR10b, Os04g10010, OS06g37224, Os06g13390, Os11g02100, Os10g28299, Os06g35700, Os12g36830, Os11g31550, Os04g09920, Os02g36190, and homologs thereof. Enhanced resistance can thus also be determined by measuring the increased expression of a gene operably linked to a promoter of a defense-related gene including but not limited to those listed above. Measurement of such expression can be measured by quantifying the accumulation of RNA or protein expression product (e.g., using northern or western blot techniques, respectively). Defense gene promoter expression can also be determined by operably linking a reporter gene to the promoter. Reporter gene constructs allow for ease of measurement of expression from the promoter of interest. Examples of reporter genes include: β-gal, GUS (see, e.g., Jefferson et al. (1987) EMBO J 6:3901), green fluorescent protein (GFP), luciferase, and others.

VIII. Examples

Introduction

The rice PRR, XA21, confers resistance to strains of the Gram-negative bacteria Xanthomonas oryzae pv. oryzae (Xoo). Xa21 codes for a predicted cell-surface localized receptor-like kinase consisting of an extracellular LRR domain, a transmembrane domain, and a non-RD cytoplasmic kinase domain (Tunkel et al., Mol. Microbiol. 58:289 (2005); Song et al., Science 270:1804 (1995)). Because identification of the PAMP that XA21 recognizes could have significant impact toward understanding this large class of receptors, we have directed a major effort towards isolation of this molecule. The present results show that raxX is a PAMP recognized by XA21.

Previous studies, using genetic approaches, led to the identification of six Xoo genes falling into two functional classes, which are required for activation of XA21-mediated immunity (rax) (see FIG. 1). Genes in the first class, raxA, raxB, and raxC, encode a predicted membrane fusion protein (MFP), an adenosine triphosphate (ATP) binding cassette (ABC) transporter protein, and an outer membrane protein (OMP), respectively. Together these three proteins are predicted to comprise a type I secretion system (T1SS), a structure known to be involved in secretion of molecules outside of the bacterial cell (da Silva et al. (2004) Mol Plant Microbe Interact 17:593; Holland et al. (2005) Mol Membr Biol 22:29). RaxB falls into a clade of ABC-transporters that carry a proteolytic peptidase domain in their N-termini, termed C39, which cleaves a characteristic double glycine (GG) motif-containing signal peptide from substrates before secretion (Holland et al. (2005) Mol Membr Biol 22:29; Dirix et al. (2004) Peptides 25:1425). RaxABC T1SS appears to be involved in the secretion of peptides/proteins and that these substrates are processed before or during secretion. We show herein that cvaB shares overlapping function with raxB.

The second class of rax genes is involved in sulfation. raxP and raxQ, encode an ATP sulfurylase and an adenosine-5′-phosphosulfate (APS) kinase. These proteins function in concert to produce 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the universal sulfuryl group donor. This class also includes the raxST-encoded sulfotransferase. raxST resides in the same operon with raxA and raxB (raxSTAB), indicating that this group of proteins targets similar substrates and/or control a similar biological process. Strains carrying mutations in these rax genes compromise the ability of Xoo to activate XA21-mediated immunity. In support of a role for these genes in a shared biological process, raxST and other rax genes are transcriptionally regulated by cell density, where high cell density induces, and low cell density represses, gene expression (Lee et al. (2006) PNAS 103:18395).

Xoo is both a serious disease of rice, an important US crop, and is a model organism for studies of other Xanthomonads, which infects virtually all plant species. Amino acid sequence identity examination of the raxX-raxSTAB genomic region reveals that orthologs are present in the agronomically important pathogens X. oryzae pv. oryzicola (Xoc) (98% identity), X. campestris pv. vesicatoria (Xcv) (88% identity), X. axonopodis pv. citrumelo (Xac) (88% identity) and X. campestris pv. musacearum (Xcm) (>85% identity). In addition, the XA21 pattern recognition receptor (PRR) class of receptors is widespread in plants and critical for plant health. The present results can thus be applied to other pathogen-host systems.

Example 1: RaxST is a Functional Tyrosine Sulfotransferase

Tyrosine sulfation was previously thought to be a process restricted to eukaryotes. Xoo RaxST is, however, capable of in vitro tyrosine sulfotransferase activity (Han et al. (2012) Nature Comm 3:1153). A raxST insertion mutant strain can overcome XA21-mediated resistance and form long lesions on XA21 carrying plants, compared with the short lesions observed with its wild type parent strain (PXO99A) (da Silva et al. (2004) Mol Plant Microbe Interact 16:593). We have recently generated a new RaxST mutant, which has the entire raxST gene deleted (PXO99AΔraxST). PXO99AΔraxST has the same phenotype as the original raxST insertion mutant (FIG. 2), further confirming that raxST is required for activation of XA21-mediated immunity.

We complemented the PXO99AΔraxST mutant phenotype by insertion of the full-length raxST driven by its putative native promoter in to the bacterial chromosome. To create this strain, we isolated the raxST gene plus a 500 bp upstream sequence and ligated it into the suicide vector pJP5603, which contains a kanamycin resistance marker. The vector also carries a 500 bp sequence that is homologous to a region of the Xoo genome located 2 Mbp upstream of the raxSTAB operon.

PXO99A, PXO99AΔraxST, and the complemented raxST deletion mutant (PXO99AΔraxST(RaxST)) were tested for their ability to form lesions on susceptible rice cultivar Tapei 309 (TP309) using an established leaf clipping method, and the resistant line expressing the XA21 receptor driven by the native promoter (TP309-XA21). FIG. 2 shows that raxST restores XA21-mediated immune activation to the PXO99AΔraxST strain. A similarly constructed mutant carrying a raxST variant with the conserved residue Arg35, part of the predicted PAPS binding motif, mutated to alanine, cannot complement the activation phenotype. In other words, Arg35 is critical for activation of XA21-mediated immunity, suggesting that PAPS binding is required for activation. All strains grow to high levels in control TP309 plants, lacking XA21.

Example 2: raxB/cvaB Double Insertion Mutant Overcomes XA21 Mediated Immunity

The presence of predicted components of a T1SS together with RaxST in the same operon led us to hypothesize that the targets of RaxST-mediated sulfation are secreted through this system. However, PXO99AΔraxST forms longer disease lesions on TP309-XA21 plants than raxA or raxB insertion mutants, indicating different effects. We recently identified a RaxB homolog, named CvaB, with 50% identity to RaxB, and created an insertion mutant in the gene encoding this protein. We found that while a mutation in cvaB alone did not alter Xoo virulence on XA21 plants, a strain carrying mutations in both raxB and cvaB resulted in significantly longer lesions than those of the raxB single insertion mutant (FIG. 3). Because CvaB is the only predicted ortholog for RaxB, we hypothesize that the raxB/cvaB double mutant is incapable of secreting proteins that activate XA21-mediated immunity.

Example 3: Identification of a Small Peptide (RaxX) that is a Candidate for RaxST-Mediated Sulfation

We hypothesized that the coding sequence for the substrate of the raxSTAB operon may be localized in the same genomic region. To identify candidates that may be substrates of RaxA/B and serve to activate XA21-mediated immunity, we created three strains carrying various deletions in the regions upstream and downstream of the raxSTAB operon. One mutant strain, carrying a 1 kb deletion upstream of RaxSTAB (PXO99ΔΔUps), could overcome XA21-mediated immunity, similar to strains carrying a deletion in raxST (FIG. 4).

We next examined whether this 1 kb region contained ORFs encoding potential targets of RaxST sulfation. We identified one small ORF, encoding a 60 amino acid polypeptide within this region called raxX, which is expressed in Xoo. All Xoo strains that carry raxX activate XA21-mediated immunity.

To test the role of raxX in XA21-mediated immunity, we complemented the PXO99AΔUps mutant with the raxX ORF plus its native promoter using the broad host range vector pVSP61. FIG. 4 shows that introduction of raxX in the PXO99AΔUps (PXO99AΔUps(raxX)) mutant partially complemented the mutant phenotype (i.e. complementation partially restored the ability of Xoo to activate XA21-mediated immunity). To confirm that raxX is responsible for the complementation, we mutated the start codon in the complementation construct (PXO99AΔUps(raxX atg-ccc)). This mutant still caused long lesions on TP309-XA21 indicating that raxX encodes a factor required for activation of XA21-mediated immunity.

Example 4: The Conserved Tyrosine of RaxX is Required for Activation of XA21-Mediated Immunity

We hypothesized that RaxX is a target of RaxST sulfation and that sulfation of RaxX is required for activation of XA21-mediated immunity. RaxX contains a tyrosine that is conserved among all of the sequenced Xanthomonas species that contain the RaxSTAB operon. Although this tyrosine is not predicted to be sulfated by three common sulfation prediction software programs, which all have been trained with eukaryotic protein sulfation sites, it does have an acidic residue in the −1 position, which is observed in some tyrosine sulfation sites. To test if this tyrosine is the target of sulfation, we generated a complementation construct encoding RaxX with the tyrosine mutated to phenylalanine (PXO99AΔUps(raxX Y41F)). The construct was introduced into PXO99AΔups, and tested for its ability to induce resistance on TP309-XA21 plants. PXO99AΔUps(raxX Y41F) formed long lesions on TP309-XA21 plants similar to the PXO99AΔUps (FIG. 4). The data thus show that RaxX is a RaxST target and suggests that sulfated RaxX is detected by XA21.

Example 5: The RaxX Deletion Mutant Overcomes XA21-Mediated Immunity

The 1 kb deletion analysis and complementation results suggest that the peptide RaxX, encoded upstream from raxST is required for activation of XA21-mediated immunity. To confirm these results, we made a more precise deletion mutant deleting only the raxX gene using a marker free approach and the suicide vector pUFR80. We complemented the marker free raxX deletion mutant with our raxX complementation construct. The mutant and complemented strain were tested for their ability to form lesions on TP309 and TP309-XA21 plants. The PXO99AΔraxX formed long lesions on TP309-XA21 (21.8 cm), whereas the complemented strain PXO99AΔraxX(raxX) caused short lesions (6.8 cm) similar to PXO99A (6.6 cm).

Example 6: Construction of a pRaxST-GFP Reporter Strain

To further investigate the role of RaxST in XA21-mediated immunity, we established a promoter-reporter system to perform a genetic screen to identify regulators of raxST. The reporter construct (pRaxST-GFP) has the raxST promoter region (defined to be −135 to −151 bp) fused to GFP (green fluorescent protein). Using qPCR, we found that expression levels of the pRaxST-GFP transcript in the PXO99A(pRaxST-GFP) strain at high cell densities is similar to levels of raxST expression in the PXO99A wild type strain. Because raxST is expressed in a cell density dependent manner in the wild-type strain, mutants that no longer express pRaxST-GFP at high cell density are likely to be impaired in the function of genes that serve as regulators of raxSTAB. Mutants in the PXO99(pRaxST-GFP) strain can thus be used to identify such regulators.

Example 7: Generation of High Purity Non-Sulfated and Sulfated RaxX-his in E. coli

To further test whether raxX is the target of RaxST-mediated sulfation and RaxAB-mediated processing and secretion, we designed a novel and efficient method to synthesize selectively sulfated proteins in E. coli. To carry out these experiments we created a RaxX fusion protein with an N-terminal MBP-tag followed by a 3C protease cleavage site and a C-terminal 6×His tag (MBP-3C-RaxX-His). MBP-3C-RaxX-His was expressed at high levels in E. coli at 20° C. overnight. MBP-3C-RaxX-His was purified on a Ni-NTA column and the N-terminal MBP-tag removed by overnight 3C protease treatment. We further purified RaxX-His by size-exclusion chromatography to homogeneity. Following this two-step purification method we are able to obtain approximately 3 mg of pure RaxX-His per liter of culture.

In order to produce sulfated RaxX-His we used a recombinant expression system for sulfated proteins where the codon for the desired sulfotyrosine residue is mutated to the TAG amber codon, which typically leads to translation termination. In this system, however, a specifically engineered orthogonal aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pair is co-expressed from the pULTRA_SYP29 plasmid. This tRNA recognizes the amber codon and incorporates sulfotyrosine, which is supplied in the media, at the desired position (Liu and Schultz (2006) Nature Biotech. 24:1436; Liu et al. (2009) Nature Protocols 4:1784). Using this system and our previously established expression and purification conditions described above for MBP-3C-RaxX-His we obtain approximately 1 mg of pure sufated RaxX(sY)-His per liter of culture. We confirmed tyrosine sulfation of RaxX(sY)-His by band shift assays on Tris-Tricine gels and selective reaction monitoring mass-spectrometry on trypsin digested peptides (FIGS. 5A and 5B).

Example 8: Purified, Sulfated RaxX Activates XA21-Mediated Immunity

Using the RaxX-His peptides purified as described above, we tested for RaxX-His and RaxX(sY)-His ability to induced defense gene expression in wild type plants or plants expressing XA21. As shown in FIG. 6, RaxX-His induced defense gene expression, e.g., PR10b, in a tyrosine sulfation and XA21-dependent manner.

Example 9: Characterization of Fragment of raxX that Activates XA21-Mediated Immunity

Five different proteases were used individually to digest 50 ug RaxX-sY (amino acids 21-60 of the full length RaxX). The digests were incubated overnight with 1 μg protease: GluC, Trypsin, ArgC, LysC, or AspN. Digest of RaxX-sY was complete for all digests when assessed by SDS-PAGE.

Detached leaf assays (Kitaake) were performed with a 500 nM (FIG. 7A) or 1 μM (FIG. 7B) peptide and gene expression was measured by qPCR after 24 hours of treatment. Controls included undigested RaxX-sY, RaxX (unsulfated), and untreated. Expression of Os06g13390 is shown as an indication of XA21-mediated gene expression.

The central ArgC peptide (amino acids 32-55) was active in these assays, indicating this fragment binds XA21 and activates pathogen resistance response. The digestion sites for each of the indicated proteases is shown in FIG. 7C.

Example 10: Establishment of Characterization Models

The raxB/cvaB double insertion mutant can be used to identify proteins that are no longer secreted in this strain. We therefore established methods to extract Xoo supernatants and characterize proteins present in this fraction using LC-MS/MS-based shotgun proteomics. To generate the supernatants, we grew Xoo cells overnight in two types of rich nutrient media (PSB and YEB) and one type of minimal nutrient media (M9) to an OD₆₀₀ of 0.5. We used three types of media to obtain a representative profile of secreted Xoo proteins that are not biased by specific growing conditions. Proteins were precipitated using 80% acetone precipitation or 0.1% TCA; trypsin and/or lysyl endopeptidase digested (to obtain maximum peptide coverage), and purified/de-salted over a C18 column prior to mass spectrometry analysis. Samples were then analyzed by LC-MS/MS and data interpreted.

Essentially, protease digested samples (˜5 μg) were analyzed by ESI-Q-TOF (Agilent 6550 iFunnel Q-TOF LC/MS). Data acquired and exported using MassHunter (Agilent Technologies) and the resultant MS/MS data was analyzed using Mascot (Matrix Sciences) against a custom database comprising the RefSeq PXO99A proteins (ca. 5000 proteins) and all Viridiplantae proteins (ca. 565,000 proteins) available through NCBI. Thresholds were also set to reduce the false discovery rate (p<0.05) and ensure significant peptide and protein matching. This led to the identification of over 100 secreted proteins from the three conditions in two independent experiments.

In addition, we successfully identified native RaxX protein in lysed cell samples of the wild type PXO99A Xoo strain. Briefly, total protein was extracted from PXO99A grown on solid YEB agar using Tris:Urea and treated similar to the methods described above. Digested peptide samples were then tested in the Orbitrap Fusion (Thermo Fisher), leading to the detection of native RaxX protein.

We have also established methods to isolate proteins from Xoo apoplastic fluids. Four week-old rice plants were infected with PXO99A using established methods (Kauffman et al. (1973) Plant Dis Rep 57:537). Approximately 100 leaves were inoculated with each strain. Six days post inoculation, 5 cm leaf sections closest to the infection site were harvested, shredded into 2 mm pieces, submerged in water and vacuum infiltrated at 22 psi four times for 15 min each. Infiltrated leaves were then surface dried and placed in a 35 ml syringe fitted into a 50 ml Falcon tube. The fitted tubes were centrifuged for 10 min at 5000 rpm twice. Approximately 7 ml of apoplastic fluid was collected from the 100 leaf samples. This strategy can be used to identify Xoo proteins that are secreted only in the presence of the host using LC-MS/MS based proteomics.

Summary

The present results demonstrate the role of the sulfotransferase RaxST in activation of the rice XA21 receptor. The results also allow us to identify genes that regulate raxSTAB expression and further characterize raxX, RaxST, RaxB, the interaction with the rice XA21 PRR, and the implications for homologous interactions. While the present results show that raxX activates XA21-mediated immunity, we cannot rule out that there are other raxST targets and that these may also activate XA21-mediated immunity.

We found a number of RaxX homologs in other Gram negative bacteria. Exemplary coding sequences are shown below in the informal sequence listing. The percent identity is shown for the amino acid sequences in FIG. 8, and the nucleotide coding sequences in FIG. 9. An alignment of raxX amino acid sequences is shown in FIGS. 10A and 10B.

Example 11

Plasma membrane localized receptors are critical components of the innate immune responses of animals and plants (Jones J D & Dangl J L (2006) Nature 444(7117):323-329; Ronald P C & Beutler B (2010) Science 330(6007):1061-1064; Kawai T & Akira S (2010) Nature immunology 11(5):373-384; Schwessinger B & Ronald P C (2012) Annual review of plant biology 63:451-482). Many of these receptors recognize and respond to the presence of conserved microbial molecules and are often referred to as pattern recognition receptors (PRRs). In animals, this recognition is carried out, in part, by toll-like receptors (TLRs) (Kawai T & Akira S (2010) Nature immunology 11(5):373-384). Humans have 10 characterized TLRs that recognize conserved microbial molecules such as lipopolysaccharide or flagellin (Poltorak A, et al. (1998) Science 282(5396):2085-2088; Hayashi F, et al. (2001) Nature 410(6832):1099-1103).

In plants, cell surface receptor kinases (RKs) and receptor-like proteins (RLPs) recognize (conserved) microbial molecules in the apoplast (Song W Y, et al. (1995) Science 270(5243):1804-1806; Chen X, et al. (2006) Plant J 46(5):794-804; Sun X, et al. (2004) Plant J 37(4):517-527; Gomez-Gomez L & Boller T (2000) Mol Cell 5(6):1003-1011; Macho A P & Zipfel C (2014) Mol Cell 54(2):263-272; Cao Y, et al. (2014) eLife 3; Hayafune M, et al. (2014) Proc Natl Acad Sci USA 111(3):E404-413). Well-characterized LRR-RKs include Arabidopsis FLS2 that detects flagellin, or its peptide epitope flg22, and the elongation factor Tu receptor (EFR) that detects the bacterial elongation factor Tu, or its peptide epitope elf18 (Gomez-Gomez L & Boller T (2000) Mol Cell 5(6):1003-1011; Zipfel C, et al. (2006) Cell 125(4):749-760). Lacking an adaptive immune system, plants have an extended array of innate immune receptors encoded in their genome. Rice, for example, has over 300 RKs predicted to serve as innate immune receptors based on the presence of a “non-RD” kinase domain which lack the arginine-aspartate motif (Dardick C & Ronald P (2006) PLoS pathogens 2(1):e2; Dardick C, Schwessinger B, & Ronald P (2012) Current opinion in plant biology 15(4):358-366). Of the few non-RD RKs characterized to date, all have a role in innate immunity or symbiosis (Schwessinger B & Ronald P C (2012) Annual review of plant biology 63:451-482; Berrabah F, et al. (2014) The New phytologist 203(4):1305-1314).

The rice XA21 RK, one of the first innate immune receptors isolated, mediates recognition of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of a serious disease of rice (Song W Y, et al. (1995) Science 270(5243):1804-1806; Khush G S, Bacalangco E, & Ogawa T (1990) Rice Genetics Newsl. 7:121-122). Previous efforts to identify the microbial molecule that activates the XA21-mediated immune response led to the identification of a number of Xoo genes that are required for activation of XA21 (rax genes). These genes encode a tyrosine sulfotransferase, RaxST, and three components of a predicted type 1 secretion system (T1SS): a membrane fusion protein, RaxA; an ATP-binding cassette transporter, RaxB; and an outer membrane protein, RaxC (da Silva F G, et al. (2004) Mol Plant Microbe Interact 17(6):593-601). raxST, raxA, and raxB are located in a single operon (raxSTAB) (da Silva F G, et al. (2004) Mol Plant Microbe Interact 17(6):593-601). Based on these findings, we hypothesized that the activator of XA21-mediated immunity is a tyrosine sulfated, type 1-secreted protein (da Silva F G, et al. (2004) Mol Plant Microbe Interact 17(6):593-601). However, the identity of this molecule has remained elusive (Lee S W, et al. (2013) Science 342(6155):191; Ronald P (2013) Scientific American; Bahar O, et al. (2014) PeerJ 2:e242). Here we report the identification of the tyrosine sulfated protein RaxX, as the activator of XA21-mediated immunity.

Results

RaxX is Required for Activation of XA21-Mediated Immunity

RaxB belongs to a subgroup of T1SS ABC transporters that contain a C39 peptidase domain (da Silva F G, et al. (2004) Mol Plant Microbe Interact 17(6):593-601). In other bacterial species, substrates for this class of secretion systems are often encoded in or near the T1SS operon (Michiels J et al., (2001) Trends Microbiol 9(4):164-168). We therefore hypothesized that the RaxST/A/B substrate may be genetically linked to the raxSTAB operon. To test this hypothesis, we generated mutants in the Xoo strain PXO99 (Salzberg S L, et al. (2008) BMC genomics 9:204) with either a 1 kb deletion ˜0.4 kb upstream from raxST (PXO99Δ1.0^(Sp)) or a 1.7 kb deletion immediately after raxB (PXO99Δ1.7^(Sp)) (FIG. 11A). The mutants were used to inoculate Taipei 309 (TP309), a rice variety lacking XA21, and XA21-TP309, a transgenic derivative expressing XA21 under control of its native promoter (Song W Y, et al. (1995) Science 270(5243):1804-1806). On TP309, all strains formed long, water-soaked lesions typical of the disease (FIG. 11B). PXO99Δ1.7^(Sp) formed short lesions on XA21-TP309 similar to the control strain PXO99. In contrast, PXO99Δ1.0^(Sp), formed long lesions on XA21-TP309. These results suggested that the region upstream to the raxSTAB operon contains an element that is required for activation of XA21-mediated immunity.

Sequence analysis of this 1 kb region revealed the presence of a 183 nucleotide ORF (covering the PXO99 genomic region 1,282,744-1,282,926). The ORF, which we named raxX (FIG. 11A), is not annotated in the PXO99 genome. We found that raxX is conserved among all sequenced Xanthomonas strains that encode the raxSTAB operon (FIGS. 15A-15B), but that it is not present in Xanthomonas species that lack the raxSTAB operon. RaxX shares no homology with other microbial proteins of known function.

To further investigate the role of RaxX, we transformed PXO99Δ1.0^(Sp) with a broad-range host vector expressing raxX under control of its native promoter (praxX). PXO99Δ1.0^(Sp)(praxX) regained the ability to activate XA21-mediated immunity and formed short lesions on XA21-TP309 (FIG. 11B). To confirm the importance of raxX in activation of XA21-mediated immunity, we generated a marker-free PXO99 mutant with the raxX open reading frame deleted (PXO99ΔraxX). As observed for PXO99Δ1.0^(Sp), PXO99ΔraxX evades the XA21-mediated immune response, forming long lesions on XA21-TP309 (FIGS. 11B, C). PXO99ΔraxX(praxX) also regained the ability to activate XA21-mediated immunity (FIGS. 11B, C). Similar results were obtained when these strains were inoculated on rice plants overexpressing XA21 in the Kitaake genetic background (Ubi::Xa21) (Park C J, et al. (2010) Plant science: an international journal of experimental plant biology 179(5):466-471) (FIGS. 12A-12D). A variant of praxX carrying a mutation in the predicted start codon (praxX-atg>tga) failed to complement PXO99ΔraxX (FIG. 11B). PXO99ΔraxX bacterial populations accumulated to more than 30-fold higher levels in inoculated XA21-TP309 rice leaves than those of strains PXO99 and PXO99ΔraxX(praxX) (FIG. 11D). These results indicate that RaxX is required for activation of XA21-mediated immunity.

RaxX is Sulfated on its Central Tyrosine by RaxST

In addition to raxST, we have previously identified two other rax genes involved in microbial sulfation. These genes, raxP and raxQ, encode an ATP sulfurylase and an adenosine-5′-phosphosulfate kinase, and work in concert to produce the universal sulfuryl group donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) (Shen Y et al. (2002) Mol Microbiol 44(1):37-48). The requirement of these three genes for activation of XA21-mediated immunity by Xoo suggests that tyrosine sulfation plays a key functional role in this process.

To further investigate this possibility, we transformed a raxST mutant strain (PXO99ΔraxST^(Sp)), which forms long lesions on XA21-TP309, with a plasmid expressing raxST under control of its native promoter (praxST). PXO99ΔraxST^(Sp)(praxST) regained the ability to activate XA21-mediated immunity. RaxST carries a predicted PAPS binding motif conserved in mammalian sulfotransferases including the human tyrosine sulfotransferases TPST1 and TPST2 (FIGS. 13A-13D) (da Silva F G, et al. (2004) Mol Plant Microbe Interact 17(6):593-601). In TPST2, mutation of the conserved arginine in the PAPS binding motif impairs enzymatic activity (Teramoto T, et al. (2013) Nat Commun 4:1572). We generated a similar mutation in raxST and tested if the expression of this mutant variant on a plasmid (praxST-R35A) could complement the PXO99ΔraxST^(Sp) infection phenotype on XA21 expressing rice plants. The strain PXO99ΔraxST^(Sp) (praxST-R35A) failed to activate XA21-mediated immunity (FIG. 12A) suggesting that sulfotransferase activity of RaxST is critical for its function.

Based on the genetic association of raxX with the raxSTAB operon, the importance of tyrosine sulfation for activation of the XA21-mediated immune response, and the presence of a single tyrosine residue in PXO99 RaxX (Y41) that is conserved between all available RaxX sequences (FIGS. 15A-15B), we hypothesized that RaxX Y41 is sulfated by RaxST and that this sulfation is required for RaxX function. To test this hypothesis, we transformed PXO99ΔraxX with a plasmid carrying a derivative of RaxX with tyrosine 41 mutated to phenylalanine (PXO99ΔraxX(praxX-Y41F)). PXO99ΔraxX(praxX-Y41F) failed to activate XA21-mediated immunity in XA21-TP309 (FIG. 12A and FIGS. 18A-18B). We also demonstrated that sulfated RaxX peptides, but not peptides carrying an Y41 to F substitution, are immunogenic on XA21 expressing rice plants (FIG. 19). These results support the hypothesis that sulfation of RaxX Y41 is required for its activation of XA21-mediated immunity.

To determine if RaxX Y41 is sulfated by RaxST in vitro, we incubated a chemically synthesized peptide covering the C-terminal region of RaxX (RaxX39, amino acids 22-60) with purified His-RaxST (FIG. 20) in the presence of PAPS. Trypsin-digested peptides were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in both negative and positive nanoelectrospray modes with ultraviolet photodissociation (UVPD) to generate informative a, b, c, x, y, and z product ions from cleavage of the peptide backbone. This method has previously been shown to facilitate the characterization of sulfated tyrosine residues within peptides by tandem mass spectrometry (MS/MS). The lability of the sulfation modification proves problematic for other MS/MS methods (Han S W, et al. (2012) Nat Commun 3:1153; Robinson M R, Moore K L, & Brodbelt J S (2014) Journal of the American Society for Mass Spectrometry 25(8):1461-1471). In negative ion mode, the sulfate group is retained on all product ions, thus allowing the sulfate modification to be unequivocally localized to Y41 of RaxX. MS/MS data showed fragment ions that account for 93% sequence coverage of peptide HVGGGDsYPPPGANPK (SEQ ID NO:4; FIG. 12B). The high-resolution verification of the peptide mass in the negative mode MS1 (HVGGGDsYPPPGANPK; SEQ ID NO:4) is displayed in FIG. 21. The extracted ion chromatograms of the peptides of interest and positive mode UVPD mass spectrum are shown in FIG. 22 and FIG. 23, respectively.

We next tested if RaxX is sulfated in vivo. Using selected reaction monitoring mass-spectrometry (SRM-MS) we observed sulfation on tryptic peptides covering Y41 derived from RaxX-His purified from PXO99(praxX-His) (FIG. 12C, FIGS. 24 and 26A-26B). The sulfated version of the tryptic peptide covering Y41 of RaxX-His purified from PXO99 ΔraxST(praxX-His) was not detectable with multiple SRM transitions above the background noise level (˜50-100 cps) (FIG. 12C, FIGS. 26A-26B). In contrast, we did detect the corresponding nonsulfated peptide covering Y41 at high levels and with high confidence for RaxX-His isolated from both PXO99 (praxX-His) and PXO99ΔraxST(praxX-His) (FIG. 12D, FIGS. 25A-25D). In combination these results demonstrate that RaxST sulfates RaxX on Y41 in vivo and that sulfation of RaxX is required for its immunogenic activity on XA21 expressing rice plants.

RaxX is Sufficient for Activation of XA21-Mediated Immunity

Infection assays using bacterial mutants clearly demonstrate that RaxX is required for activation of XA21-mediated immunity. We next sought to determine if sulfated RaxX can trigger XA21-mediated defense responses in the absence of Xoo. For this purpose, we produced full-length sulfated recombinant RaxX (RaxX60, 1-60 aa) using an expanded genetic code approach (Liu C C & Schultz P G (2006) Nature biotechnology 24(11):1436-1440; Lajoie M J, et al. (2013) Science 342(6156):357-360). We heterologously expressed RaxX in E. coli together with an engineered aminoacyl-tRNA synthetase specific for sulfotyrosine, a cognate engineered amber suppressor tRNA and the non-standard amino acid sulfotyrosine (sY) (Liu C C et al., (2009) Nature protocols 4(12):1784-1789). Nonsulfated RaxX was also expressed in E. coli without sulfotyrosine. We confirmed purity and tyrosine sulfation status of RaxX60 by gel-based assays and SRM-MS analysis (FIG. 25A-25D). We tested if the resulting highly purified, full-length, sulfated RaxX60-sY and nonsulfated RaxX60-Y proteins could trigger defense gene expression in leaves of rice plants overexpressing XA21 (Ubi::XA21). As shown in FIGS. 27A-27B, sulfated RaxX60-sY, but not the nonsulfated form RaxX60-Y, triggered strong up-regulation of defense marker genes (Table 3) in detached leaves of Ubi::XA21. Leaves from Kitaake rice plants, which lack the XA21 immune receptor, are insensitive to RaxX60-Y and RaxX60-sY. These results demonstrate that sulfated RaxX60-sY is sufficient to activate XA21-mediated defense gene expression in rice.

To identify a ‘minimal’ epitope of RaxX that is sufficient to trigger these responses, we took biochemical and rational design approaches. We first tested whether chemically synthesized RaxX39 is sufficient to trigger XA21-dependent defense gene expression (FIG. 13A, FIGS. 28A-28B). We found that sulfated RaxX39-sY triggers defense gene expression in an XA21-dependent manner, whereas nonsulfated RaxX39 does not (FIGS. 28A-28B). To further narrow down the active region, we subjected RaxX39 to digestion with four site-specific proteases (GluC, ArgC, AspN, and trypsin). The predicted digestion patterns were confirmed by gel-based assays and by SRM-MS analysis for ArgC, AspN and trypsin digests (FIGS. 29A-29D). We tested the resulting RaxX fragments for their ability to activate XA21-dependent signaling (FIG. 30A). Only RaxX39-sY digestion products resulting from GluC and ArgC treatments retained activity on Ubi::XA21 plants. The ability of the ArgC fragment (32-55 aa) to activate XA21-mediated immunity was confirmed with chemically synthesized RaxX24-sY (32-55 aa) peptides (FIG. 13A, FIG. 30B). Next, we tested N- and C-terminal truncated versions of RaxX24-sY peptides. Chemically synthesized RaxX21-sY (35-55 aa) retained the ability to induce XA21-dependent signaling whereas RaxX18-sY (32-49 aa) was compromised in this activity (FIG. 13A, FIG. 30B). These results indicate that a chemically synthesized tyrosine sulfated 21 amino acid derivative of RaxX, named RaxX21-sY, is sufficient to activate XA21-mediated defense responses.

In addition to activation of defense marker gene expression, the activation of PRR-triggered immunity in plants often involves the production of ethylene and reactive oxygen species (ROS) (Macho A P & Zipfel C (2014) Mol Cell 54(2):263-272; Boller T & Felix G (2009) Annual review of plant biology 60:379-406). These responses are known to contribute to the final disease outcome in many plant pathogen interactions (Macho A P & Zipfel C (2014) Mol Cell 54(2):263-272; Boller T & Felix G (2009) Annual review of plant biology 60:379-406). We therefore tested if RaxX21-sY is able to trigger these hallmarks of plant innate immune signaling in XA21-expressing rice leaves (FIGS. 13B-13D, FIGS. 31A through 33B). As shown in FIGS. 13B-13D, only sulfated RaxX21-sY, but not RaxX21-Y, was able to activate defense marker gene expression and the production of ROS and ethylene in an XA21-dependent manner. These responses were most pronounced in rice plants overexpressing XA21 (Ubi::XA21). Nonetheless, we also observed significant RaxX21-sY-mediated upregulation of defense gene expression and ROS production in Kitaake lines expressing XA21 from its native promoter (XA21::XA21) (FIGS. 32 through 33B) with the highest levels of ROS production observed in rice leaves harvested at later stages of development (FIGS. 33A-33B).

To assess the sensitivity of XA21-expressing rice plants towards the different RaxX epitopes, we established the RaxX concentration needed to trigger the half maximal response (often referred to as the EC50 value) using the fast and quantitative ROS assay. The EC50 value in Ubi::XA21-expressing rice plants ranges from ˜100 nM for RaxX60-sY, ˜70 nM for RaxX39-sY to ˜30 nM for RaxX24-sY and ˜20 nM for RaxX21-sY (FIG. 13A and FIGS. 34A-34E). In contrast, the further truncated peptide RaxX18-sY was completely inactive, even at high peptide concentrations (FIGS. 34A-34E). As an additional control, we tested an unrelated tyrosine sulfated peptide called axY(S)22, derived from a PXO99 outer membrane protein (ACD57244), for its ability to activate XA21-mediated immunity (Lee S W, et al. (2013) Science 342(6155):191; Bahar O, et al. (2014) Peer J 2:e242). Rice leaves over-expressing XA21 were insensitive to axY(S)22 at high peptide concentrations (1 μM) under conditions in which RaxX21-sY induced ROS production and defense gene expression changes (FIGS. 35A-35B). In summary, our data demonstrates that XA21-mediated immune signaling in rice is specifically induced by RaxX60-sY, and sulfated epitopes derived from the full RaxX60 protein, at low nanomolar concentrations.

Identification of a RaxX Variant that Evades XA21-Mediated Immunity

When XA21 was first discovered, it was demonstrated to confer resistance to most tested strains of Xoo (Khush G S, Bacalangco E, & Ogawa T (1990) Rice Genetics Newsl. 7:121-122; Wang G L et al. (1996) Mol Plant Microbe Interact 9(9):850-855). Since that time, several field strains that evade XA21-mediated immunity have been identified (Mishra D, et al. (2013) PLoS One 8(11):e81996; Wang C L, et al. (2014) Mol Plant Pathol 15(4):333-341). For most of these strains, the molecular basis of immune evasion is unknown. However, we previously demonstrated that two of these Xoo strains, DY89031 and CK89021, carry mutations in raxA and raxST, respectively (da Silva F G, et al. (2004) Mol Plant Microbe Interact 17(6):593-601). We hypothesized that other strains may evade XA21-mediated immunity because they encode variant raxX alleles. Indeed, we found that RaxX from the Indian field isolate IXO685, which evades XA21 mediated immunity (Mishra D, et al. (2013) PLoS One 8(11):e81996), differs in 9 amino acids as compared with RaxX from PXO99 (FIG. 14A). Four amino acid changes are located in the RaxX21 sequence.

We tested if raxX from IXO685 expressed under control of the native promoter of PXO99 raxX was able to complement the PXO99ΔraxX mutant phenotype on XA21 expressing rice plants. We found that PXO99ΔraxX(praxX-IXO685) was unable to activate XA21-mediated immunity (FIG. 14B). Likewise, recombinant, highly purified RaxX60-sY-IXO685 produced using our expanded genetic code system was not able to trigger XA21-mediated ROS production (FIG. 14C, FIG. 36). Expression of the PXO99 raxX allele in the IXO685 strain conferred the strain with the ability to activate XA21-mediated immunity (FIG. 14B). These results indicate that IXO685 is able to overcome XA21-mediated immunity because it has acquired several non-synonymous substitutions in the raxX ORF. Two other Indian field isolates, (IXO651 and IXO1221), which are also able to evade XA21-mediated immunity (Mishra D, et al. (2013) PLoS One 8(11):e81996) and share the identical raxX sequence with IXO685 can also be complemented with prax (FIG. 37).

To determine if the four identified residues within the RaxX21 minimal epitope that differ with the XA21-evading strains are essential for XA21 activation, we carried out mutational analyses. The four residues were mutated in praxX and the mutated alleles were tested for functional complementation of the PXO99ΔraxX mutant by inoculation of XA21-expressing rice plants (FIG. 14D). PXO99ΔraxX(praxX-P44S) and PXO99ΔraxX(praxX-P48T) formed long lesions on XA21-TP309; PXO99ΔraxX(praxX-A46P) displayed an intermediate phenotype; and PXO99ΔraxX(praxX-R55P) formed short lesions. Consistent with this result, highly purified recombinant RaxX60-sY-P44S and RaxX60-sY-P48T proteins, produced using our expanded genetic code system, are unable to induce ROS production in Kitaake Ubi::XA21 (FIG. 14E, FIG. 36). Thus, P44 and P48 are required for activation of XA21-mediated immunity. In other words, the alterations of these specific amino acids in RaxX of IXO685, IXO651, and IXO1221 enable these strains to evade XA21-mediated immunity.

Discussion

Our results indicate that the presence or absence of sulfation and the residues near Y41 are decisive for the ability of RaxX to trigger XA21-mediated immunity and that Y1 is a substrate for RaxST-mediated sulfation. We have identified RaxX in at least 8 Xanthomonas species that infect maize, cassava, sugar cane, tomatoes, peppers, wheat, alfalfa, onions, banana, and citrus (FIGS. 11A-11D). In all of these strains, raxX is encoded upstream of raxST, raxA, and raxB. The colocalization of the rax genes suggest that they function in a common biological process and that the RaxX proteins in these species are also substrates for RaxST-mediated sulfation.

While tyrosine sulfation has not been demonstrated in other prokaryotic species, it is a common posttranslational modification of eukaryotic proteins and plays important roles in regulating plant development and in mediating the interactions of plants and animals with microbes. For example, in humans, sulfation of the C—C chemokine receptor type 5 (CCR5) is critical for binding of the envelope glycoprotein gp120 by the human immunodeficiency virus (HIV) (Farzan M, et al. (1999) Cell 96(5):667-676). In plants, the sulfated peptide signaling factors PSK (phytosulfokine) and PSY1 (plant peptide containing sulfated tyrosine 1) have been implicated in xylem trachea development, root growth, suppression of plant defense and promotion of plant health (Matsubayashi Y (2014) Annual review of plant biology 65:385-413; Mosher S, et al. (2013) Plant J 73(3):469-482).

Although RaxX shares no homology with microbial proteins of known function, we have noted a remarkable similarity between the sulfated, secreted Arabidopsis 18 amino acid peptide signaling factor PSY1, four predicted rice PSY1 orthologs and RaxX residues 40-55 (FIGS. 38A-38B). These similarities suggest the exciting possibility that in the absence of XA21, Xoo and other Xanthomonads utilize sulfated RaxX to mimic PSY-like peptides in order to suppress host defense responses, facilitate infection or enhance plant health, maintenance of which is critically important for propagation and survival of biotrophic pathogens (Mosher S, et al. (2013) Plant J 73(3):469-482; Amano Y et al. (2007) Proc Natl Acad Sci USA 104(46):18333-18338; Mosher S & Kemmerling B (2013) Plant Signal Behav 8(5):e24119).

The robust immunity conferred by XA21 to most Xoo strains is a highly valued agronomic trait and therefore XA21 has been introgressed into diverse rice varieties grown by rice farmers (Sundaram M, et al. (2008) Marker assisted introgression of bacterial blight resistance in Samba Mahsuri, an elite indica rice variety. 160 3(411-422); Huang B et al. (2012) Euphytica 187:449-459; Chen S et al. (2000) Crop Science 40(1):239-244; Toenniessen G H, O'Toole J C, & DeVries J (2003) Current opinion in plant biology 6(2):191-198; Gopalakrishnan S, et al. (2008) Plant Breeding 127(2):131-139; Win K M, et al. (2012) Field Crops Research 137:186-194). The discovery of raxX and allelic variants that can evade XA21-mediated recognition can be used to screen Xoo field populations for the presence of potentially virulent variants. Such an anticipatory breeding approach would alert farmers of the need to plant different varieties or mixtures prior to the breakdown of XA2J-mediated immunity.

The knowledge gained from these studies can also be used to expand the spectrum of XA21-mediated immunity to additional strains of Xoo. For example, new synthetic receptors of XA21 with novel engineered recognition specificities such as those that can recognize RaxX from IXO685 would be of great interest. Because Xanthomonas species affect virtually all crop plants, it is expected that future studies will lead to new strategies for engineering resistance to non-rice pathogens. For example, X. axonopodis pv. citrumelo, X. euvesicatoria and X. oryzae pv. oryzicola, which cause serious diseases on citrus, tomato, and rice, carry alternate raxX alleles (FIGS. 15A-15B). This knowledge provides an opportunity to engineer new XA21 variants that recognize these alleles and use the novel variants for engineering plants resistant to Xanthomonas infections that cannot be controlled by more conventional genetic methods.

The sulfated RaxX/Xa21 interaction represents a new biological system for studies of the interaction of sulfated epitopes and their receptors. A recent report of the generation of a potent new anti-viral drug (Gardner M R, et al. (2015) Nature 519(7541):87-91), which was based on more than 10 years of work on a single family of sulfated peptides, indicates the clinical relevance of studies of sulfopeptides. Thus the studies reported here are applicable to generation of therapeutic reagents with the potential to block microbial infection of both plants and animals.

Materials and Methods

Bacterial Culture and Growth Conditions

Lists of plasmids and bacterial strains used in this study are provided in Table 1 and S2. Xoo was cultured on peptone sucrose agar (PSA) plates at 28° C. with the appropriate antibiotic(s). YEB medium (5 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl, 5 g/L sucrose, 0.5 g/L MgSO₄, pH 7.3) was used for liquid culture at 28° C. with shaking at 230 rpm. E. coli was cultured at 37° C. and 230 rpm in lysogenic broth (LB) for cloning or M9T (per 1 liter: M9 plus 10 g tryptone, 5 g NaCl; after autoclaving add 1 mL of 1M MgSO4 and 1 mL of 0.02% biotin for expression). Kanamycin was used at 50 μg/mL, carbenicillin at 50 μg/mL, spectinomycin at 50 μg/mL, and cephalexin at 20 μg/mL.

Xoo Mutation and Complementation

Xoo mutants were generated in Philippine race 6 strain PXO99Az, a derivative of strain PXO99 (referred to simply as PXO99 in this manuscript) (Salzberg S L, et al. (2008) BMC genomics 9:204; Hopkins C M et al., (1992) Mol Plant Microbe Interact 5(6):451-459). PXO99ΔraxST^(Sp), PXO99Δ1.0^(Sp), and PXO99Δ1.7^(Sp) were generated by marker exchange mutagenesis using the suicide vector pJP5603 (Penfold R J & Pemberton J M (1992) Gene 118(1):145-146). ˜500 bp sequences flanking both sides of the region to be deleted were sequentially cloned into the multiple cloning site using the restriction enzyme sites Kpnl/BamHI and BamHI/SalI. A spectinomycin resistance cassette was ligated between the two flanking sequences using the BamHI site. PXO99 competent cells were transformed with the suicide plasmids by electroporation and plated to PSA with 50 μg/mL spectinomycin. Colonies with the expected antibiotic resistance phenotype for double crossover (spectinomycin resistant, kanamycin sensitive) were validated by PCR.

Marker free raxX (PXO99ΔraxX) and raxST (PXO99ΔraxST) mutants were generated using the vector pUFR80 (Castañeda A et al., (2005) Mol Plant Microbe Interact 18:1306-1317). ˜500 bp sequences flanking were sequentially cloned into the multiple cloning site of pUFR80 using the sites EcoRI/BamHI and BamHI/HindIII. praxX-KO-MF and praxST-KO-MF were transformed into PXO99 competent cells by electroporation, and the cells were plated to nutrient agar (NA) (CM0001, OXOID) with 50 μg/mL kanamycin. Transformants were grown in nutrient broth without kanamycin and plated to NA with 5% sucrose to select for a second crossover event. Colonies were analyzed by PCR to identify deletion mutants.

Strains were transformed using derivatives of the broad host range vector pVSP61 (Loper J E & Lindow S E (1994) Applied and environmental microbiology 60(6):1934-1941). The raxX and raxST sequences along with their predicted promoter region (300 bp upstream of the predicted start codons) were cloned into pVSP61 using the EcoRI/HindIII sites. Point mutations were generated by QuikChange mutagenesis. For praxX-IXO685, the raxX promoter from PXO99 was first introduced into pVSP61 using the sites EcoRI/BamHI to generate praxXprom. The raxX sequence from IXO685 was amplified from genomic DNA and cloned into praxXprom behind the PXO99 raxX promoter using the restriction sites BamHI/HindIII.

Rice and Rice Growth Conditions for Inoculations

Oryza sativa ssp. japonica rice varieties Taipei 309 (TP309), a 106-17-derived transgenic line of TP309 carrying XA21 driven by its own promoter (XA21-TP309) (Song W Y, et al. (1995) Science 270(5243):1804-1806), Kitaake, and Ubi::Myc::XA21-Kitaake line 7-A-8 (Ubi::XA21) (Park C J, et al. (2010) Plant science: an international journal of experimental plant biology 179(5):466-471) were used for rice inoculations. Native Tapei 309 and Kitaake do not contain XA21. Seeds were germinated in distilled water at 28° C. for one week, and then transplanted into sandy soil (80% sand, 20% peat from Redi-Gro, Sacramento, Calif.) in 5.5 inch square pots (2 seedlings/pot). Plants were grown in tubs in a greenhouse. The plants were top watered daily with fertilizer water (N, 58 ppm; P, 15 ppm; K, 55 pm; Ca, 20 ppm; Mg, 13 ppm; S, 49 ppm; Fe, 1 ppm; Cu, 0.06 ppm; Mn, 0.4 ppm; Mo, 0.02 ppm; Zn, 0.1 ppm; B, 0.4 ppm) for 4 weeks, followed by water for 2 weeks. Six weeks after planting, the rice was transferred to a growth chamber set to 28° C./24° C., 80%/85% humidity, and 14/10 h lighting for the day/night cycle.

Plants were inoculated 2-3 days after transfer using the scissors clipping method (Kauffman H et al. (1973) Plant Disease Reporter 57:537-541). Bacteria for inoculation were taken from PSA plates and resuspended in water at a density of 10⁸ colony forming units per mL. Water soaked lesions were measured 14 days after inoculation. In planta bacterial growth analysis was carried out as described (Bahar O, et al. (2014) PeerJ 2:e242.). Four biological replicates were measured for each data point. Each biological replicate was the mean number of colonies from four technical replicates.

Generation of RaxX Peptides

Sulfated (sY) and nonsulfated (Y) versions of RaxX39 (KGRPEPLDQRLWKHVGGGDYPPPGANPKHDPPPRNPGHH; SEQ ID NO:3), RaxX24 (LWKHVGGGDYPPPGANPKHDPPPR), RaxX21 (HVGGGDYPPPGANPKHDPPPR), RaxX18 (LWKHVGGGDYPPPGANPK) and ax22(S)Y (Lee S W, et al. (2013) Science 342(6155):191) were ordered from Pacific Immunology. The peptides were checked for purity by HPLC and MS analysis by the company. All peptides were resuspended in ddH₂O. No other solvents were required to dissolve the peptide.

For the expanded genetic code approach, full length RaxX (RaxX60) from PXO99 was expressed as an MBP-3C-RaxX-His fusion protein in E. coli C321. ΔA.exp (Lajoie M J, et al. (2013) Science 342(6156):357-360). E. coli C321. ΔA.exp was cotransformed with SYP29 pULTRA SY, made by cloning the sY-specific aminoacyl-tRNA synthetase from (Liu C C et al., (2009) Nature protocols 4(12):1784-1789) into the pULTRA system (Chatterjee A et al. (2013) Biochemistry 52(10):1828-1837) and pBAD/MBP-3C-RaxX-His (Amber) to generate sulfated RaxX or pBAD/MBP-3C-RaxX-His to generate nonsulfated RaxX. Transformed bacteria were grown in 0.5 L of M9T media. For expression of sulfated RaxX, sulfotyrosine (sY), synthesized as described in (Liu C C et al., (2009) Nature protocols 4(12):1784-1789), was added to a final concentration of 3 mM. Cultures were grown at 37° C. with shaking at 230 rpm. Expression was induced at an O.D. of 0.6-0.7 by addition of 1 mM IPTG and 0.25% (w/vol) arabinose for 5 h. MBP-3C-RaxX-His was purified from intracellular total protein extracts over a 5 mL Ni-NTA column on an FPLC (Akta, GE Healthcare). The MBP-tag was removed by overnight digestion with 3C protease at a concentration of 1:200 (w/w) at 4° C. The MBP was separated from full length RaxX-His over a 1 mL SP-XL column, a strong cation exchanger, on an FPLC (Akta, GE Healthcare). Nonsulfated RaxX was expressed and purified in the same way, except pBAD/MBP-3C-RaxX-His plasmids without Amber mutations were used, and no sulfotyrosine was added to the cultures. The yield for sulfated and nonsulfated highly purified RaxX-His was about 1 mg protein per 1 L of culture. All RaxX allelic variants were prepared according to the same protocol.

In Vitro Sulfation of RaxX by his-RaxST

RaxST was expressed as a full length fusion protein with an N-terminal His-tag in BL21(DE3) cells and purified using Ni-NTA agarose beads (QIAGEN). His-RaxST expression was confirmed by SDS-PAGE with Coomassie-blue staining and western blot analysis with Penta-His™ antibody (QIAGEN) (FIGS. 18A-18B). The in vitro sulfation reaction was carried out as described in Han et al (Han S W, et al. (2012) Nat Commun 3:1153). 20 μg of the purified RaxST was incubated with 170 μg of RaxX39 in the presence of sulfation buffer (25 mM Tris pH 7.5, 150 mM NaCl, 20 mM MgCl₂, 480 μM PAPS) for 4 h at 30° C.

nanoLC-UVPD Mass Spectrometry

Proteins from in vitro sulfation reactions were buffer exchanged into 50 mM Tris HCl (pH 8.5), 1 M urea using a 3 KDa molecular weight cut-off (MWCO) filter and digested with trypsin overnight at 37° C. using a 1:25 enzyme to substrate ratio. Following digestion, samples were passed through a 10 K MWCO filter to separate RaxX39 peptides from trypsin.

Tryptic peptides of RaxX39 were separated by reversed phase liquid chromatography at 300 nL/min using a Dionex Ultimate 3000 NSLC system conFIG.d for preconcentration. Mobile phases A and B were water and acetonitrile, respectively, both containing 0.1% formic acid. A solution of 2% acetonitrile and 0.1% formic acid was used for sample loading onto a New Objective IntegraFrit 100 μm×3 cm trap column (Woburn, Mass.) packed in-house with 5 μm, 100 Å Michrom Magic C18 AQ (Auburn, Calif.). After 5 min of preconcentration at 7 μL/min, the trap column was switched in-line with a with a New Objective PicoFrit analytical column (Woburn, Mass.) (75 μm×15 cm) packed with Waters 3.5 μm, 130 Å XBridge BEH C18 (Milford, Ma). A 23 min linear gradient starting at 2% B and ending at 40% B was used for separation.

LC-MS/MS analysis was performed on a Thermo Scientific Velos Pro mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Coherent (Santa Clara, Calif.) Excistar XS excimer laser operated at 193 nm with a pulse frequency of 500 Hz (Madsen J A, et al. (2013) Molecular & cellular proteomics: MCP 12(9):2604-2614). Data acquisition was carried out by first performing a full MS1 scan from m/z 300-2000 followed by 3 MS/MS UVPD events to activate the doubly charged sulfopeptide of interest, HVGGGDsYPPPGANPK (SEQ ID NO:4), at m/z 769.8 in negative mode and m/z 771.8 in positive mode. A single laser pulse was used for ion activation in negative mode during a 2.0 ms activation period. Two laser pulses were used for ion activation in the positive mode. In both polarities the isolation width was 3 Da and the activation q value was 0.10. MS/MS fragmentation patterns were manually interpreted. For the UVPD mass spectrum shown in FIG. 12B, the neutral loss of SO₃ and CO₂ from the precursor ion and/or charge reduced precursor ion are denoted as “—SO₃” and “—CO₂”, respectively in the spectrum. Product ions a+1 and x+1 correspond to homolytic cleavage of the Cα-carbonyl bond of the peptide backbone without subsequent transfer of hydrogen radicals to produce even electron a and x ions which are the diagnostic N-terminal and C-terminal ions used to identify the peptide.

High resolution mass measurement of the diagnostic tryptic peptide corresponding to HVGGGDsYPPPGANPK (SEQ ID NO:4) from the RaxX39 sample sulfated by RaxST in vitro was undertaken on a ThermoFisher Scientific Instruments Orbitrap Elite mass spectrometer operated at an FT resolution of 120,000 at m/z 400.

Isolation of RaxX from PXO99 for Selected Reaction Monitoring Mass Spectrometry

Briefly, RaxX-His proteins were purified from Xoo strains and their sulfation was analyzed by SRM-MS. PXO99(praxX-His) and PXO99ΔraxST(praxX-His) were cultured in 1 L XVM2 (Wengelnik K & Bonas U (1996) J Bacteriol 178(12):3462-3469) liquid media for 40 h at 28° C. The cells were harvested by centrifugation at 10,000 rpm for 20 min at 4° C. RaxX-His was purified from intracellular total protein extract over a 5 mL Ni-NTA column on an FPLC (Akta, GE Healthcare). Isolation of Rax-His was confirmed by Coomassie-blue staining and western blot analysis with Penta-His™ antibody (QIAGEN) (FIG. 20).

Selected Reaction Monitoring Mass Spectrometry (SRM-MS)

Purified RaxX-His was digested with 1 μg of tryp sin in the presence of 5 mM DTT. Peptide digests were analyzed using an Agilent 1260 liquid chromatography system (Agilent, Santa Clara, Calif.) operating in normal flow mode at 400 μL/min coupled to an Agilent 6460QQQ Mass Spectrometer (Agilent). Peptides were separated on an Ascentis Express Peptide C18 column (2.7 μm particle size, 160 Å pore size, 5 cm length×2.1 mm i.d., coupled to a 5 mm×2.1 mm i.d. guard column with similar particle and pore size, operating at 60° C.; Sigma-Aldrich, St. Louis, Mo.) using a 43-minute method with the following gradient: initial conditions were 98% Buffer A (0.1% formic acid), 2% Buffer B (99.9% acetonitrile, 0.1% formic acid) after which B was increased to 35% over 35.70 min, followed by an increase to 90% B in 18 s, where it was held for 2 min. Buffer B was then decreased to 5% in 30 s where it was held for 2.5 min, then further decreased to 2% and held for 2 min to re-equilibrate the column.

The eluted peptides were ionized via an Agilent Jet Stream ESI source operating in positive-ion mode with the following source parameters: Gas temperature=250° C., Gas flow=13 L/min, Nebulizer pressure=35 psi, Sheath Gas temperature=250° C., Sheath Gas flow=11 L/min, Capillary voltage=3500 V, Nozzle voltage=0 V. The data were acquired using Agilent MassHunter, version B.06.00. Data were acquired in MRM mode with transitions generated via Skyline (MacLean B, et al. (2010) Bioinformatics 26(7):966-968) software (version 2.6). In silico SRM transitions were selected with 2+ and 3+charge states, no missed tryptic cleavages, sulfated and non-sulfated tyrosine modifications, and singly-charged fragment ions from both the y- and b-series. The Skyline generated method was refined to three transitions per peptide and set to Agilent 6460QQQ for instrument-specific collision energies. The final method consists of 36 transitions, with a 25-ms dwell time per transition and MS1 and MS2 resolution set to unit. Acquired SRM data were imported into Skyline where confident peptide quantification was based on multiple concurrent SRM transitions above background signal and peak areas were refined with the mProphet algorithm (Reiter L, et al. (2011) Nat Methods 8(5):430-435).

Rice and Rice Growth Conditions for Plant Defense Assays

Kitaake, Ubi::Myc::XA21-Kitaake line 7-A-8 (Ubi::XA21) (Park C J, et al. (2010) Plant science: an international journal of experimental plant biology 179(5):466-471) and XA21::XA21-Kitaake (Park C J, et al. (2010) Plant science: an international journal of experimental plant biology 179(5):466-471) were grown in growth chambers at 24° C. or 28° C. with a 14h/10 h light-dark cycle at 80% humidity. Rice plants were grown in hydroponic system using A-OK Starter Plugs (Gordan) and watered with Hoaglands solution twice a week.

Assays for Reactive Oxygen Species

Leaves of 4- to 6-week-old hydroponically grown rice plants were cut longitudinally along the mid vein and then transversely into 1 to 1.5 mm thick leaf pieces. These leaf pieces were floated on autoclaved ddH₂O overnight. The next morning two leaf pieces were transferred into one well of a 96-well white plate containing 100 μL excitation solution (5-20 mM L-012 [Wako, Japan], 0.5-2 mg/mL HRP [Sigma]). Leaf pieces were treated with the indicated concentration of peptides or mock treated (ddH₂O or a peptide buffer solution). Chemiluminescence was measured for 3 h with 0.5 s per reading with a high sensitivity plate reader (TriStar, Berthold, Germany).

Gene Expression Assays

Rice leaf tissue was treated with peptides as described previously with slight adaptations (Chen X, et al. (2014) Molecular plant 7(5):874-892). Leaves of 4-week-old hydroponically grown rice plants were cut into 2 cm long strips and incubated for at least 12 h in ddH₂O to reduce residual wound signal. Leaf strips were treated with the indicated peptides and for the indicated time. Leaf tissue was snap-frozen in liquid nitrogen and processed appropriately. PR10b and Os04g10100 were previously described as defense marker genes in rice (Chen X, et al. (2014) Molecular plant 7(5):874-892). Other defense marker genes, Os12g36830 and Os06g37224, were identified in RNAseq experiments as upregulated genes after elf18 treatment of rice leaves expressing EFR::XA21::GFP (Schwessinger B, et al. (2015) PLoS pathogens 11(3):e1004809), (internet site genome.jgi.doe.gov/pages/dynamicOrganismDownload.jsf?organism=OrysatspilotREDO). For qRT-PCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. The genes and qRT-PCR primer pairs are listed in Table 3. qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62° C. for 5 s and denaturation at 95° C. for 5 s. The expression levels of all defense marker genes were normalized to the actin gene expression level and to the respective mock treated control at 0 or 1 h.

Ethylene Production

Leaves of 3-4 weeks old hydroponically grown rice plants were cut in small horizontal strips (˜0.3-0.5 mm) and floated on water in Petri dishes overnight at room temperature. Ethylene biosynthesis was assayed by placing leaf samples in 6-mL tubes with 500 μL of water or the indicated peptide at a concentration of 1 μM as indicated. Tubes were sealed with rubber caps, and ethylene accumulating in the headspace within 4 h of incubation was determined by gas chromatography as described (Felix G et al. (1999) Plant J 18(3):265-276).

RaxX39 Peptide Digestion

10 μg of RaxX39-Y or RaxX39-sY were digested in buffer D (50 mM Tris HCl, 1 mM DTT) with 1 μg of one of the following site-specific proteases: GluC, ArgC, AspN, or trypsin. Digestions or mock reactions were performed at 25° C. for 16 h.

Statistical Analysis

Statistical analyses were performed using JMP software (ASAS Institute Inc., Cary, N.C., USA).

TABLE 1 Plasmids used in this study. Strain Relevant characteristics Source Xoo suicide plasmids for mutant generation pJP5603 Suicide vector, requires pir+ strain for Penfold R J & replication, Km^(R) Pemberton J M (1992) Gene 118(1): 145- 146 pUFR80 Suicide vector with sacB, for double Castañeda A crossover selection on sucrose, Km^(R) et al., (2005) Mol Plant Microbe Interact 18: 1306-1317 praxST-KO-Sp pJP5603-based vector for deletion of This study raxST, contains flanking regions 1,282,801- 1,283,300* and 1,284,171-1,284,648, Km^(R)Sp^(R) pΔ1.7-KO-Sp pJP5603-based vector for deletion of This study PXO99 genomic region 1,287,700- 1,289,400, contains flanking regions 1,287,199-1,287,699 and 1,289,401- 1,289,900, Km^(R)Sp^(R) pΔ1.0-KO-Sp pJP5603-based vector for deletion of This study PXO99 genomic region 1,282,000- 1,283,000, contains flanking regions 1,281,498-1,281,999 and 1,283,001- 1,283,501, Km^(R)Sp^(R) praxX-KO-MF pUFR80-based vector for marker free (MF) This study deletion of PXO99 raxX (1,282,744- 1,282,926), contains flanking regions 1,282,254-1,282,743 and 1,1,282,927- 1,283,416, Km^(R), sacB⁺ praxST-KO-MF pUFR80-based vector for marker free (MF) This study deletion of PXO99 raxST, contains flanking regions 1,282,801-1,283,300 and 1,284,171-1,284,648, Km^(R), sacB⁺ Xoo complementation plasmids pVSP61 Broad host range vector used for Loper J E & complementation, Km^(R) Lindow S E (1994) Applied and environmental microbiology 60(6): 1934- 1941 praxST raxST with native promoter (300 bp) from This study PXO99 in pVSP61, Km^(R) praxST-R35A praxST, arginine 35 mutated to alanine, This study Km^(R) praxX raxX with native promoter (300 bp) from This study PXO99 (c1,282,744-1,283,226) in pVSP61, Km^(R) praxX-atg>tga praxX, raxX start codon mutated to stop This study codon TGA, Km^(R) praxX-Y41F praxX, tyrosine 41 mutated to This study phenylalanine, Km^(R) praxX-P44S praxX, proline 44 mutated to serine, Km^(R) This study praxX-A46P praxX, alanine 46 mutated to proline, Km^(R) This study praxX-P48T praxX, proline 48 mutated to threonine, This study Km^(R) praxX-R55P praxX, arginine 55 mutated to proline, Km^(R) This study praxX-His praxX with a 6xHis tag at the end of raxX This study praxXprom raxX promoter (300 bp) from PXO99 - This study (c1,282,927-1,283,226) in pVSP61, Km^(R) praxX-IXO685 raxX from Xoo strain IXO685 cloned behind This study the PXO99 raxX promoter (300 bp) in pVSP61, Km^(R) E. coli expression plasmids SYP29_pULTRA_SY The sY-specific aminoacyl-tRNA synthetase This study from (Liu C C et al., (2009) Nature protocols 4(12): 1784-1789) cloned into the pULTRA system (Chatterjee A et al. (2013) Biochemistry 52(10): 1828-1837), Sp^(R) pBAD/Myc-His A Cb^(R) Invitrogen pET28a(+) Km^(R) Novagen pET28a(+):raxST Full length of PXO99 raxST cloned into This study pET28a(+) between EcoRI and XhoI restriction enzyme sites, Km^(R) pMAL/MBP-3C-RaxX- raxX from PXO99 (c1,282,926-1,283,226) This study His cloned into pMAL-c4X by addition of a 3C coding sequence at the 5′-end and a 6-His coding sequence followed by a stop codon at the 3′-end, Cb^(R) pBAD/MBP-3C-RaxX- MBP-3C-RaxX-His coding for raxX from This study His PXO99 (c1,282,926-1,283,226) cloned into pBAD/Myc-His A by PCR amplification, restriction ligation using NcoI/HindIII, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His, tyrosine 41 This study Y41F-His mutated to phenylalanine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His, proline 44 This study P44S-His mutated to serine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His, alanine 46 This study A46P-His mutated to proline, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His, proline 48 This study P48T-His mutated to threonine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His, arginine 55 This study R55P-His mutated to proline, Cb^(R) pBAD/MBP-3C-RaxX- MBP-3C-RaxX-His coding for raxX from This study His (Amber) PXO99 (c1,282,926-1,283,226) cloned into pBAD/Myc-His A with a change of the Y41 codon from TAT to TAG for incorporation of the non-standard amino acid sulfotyrosine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His (Amber), tyrosine This study Y41F-His (Amber) 41 mutated to phenylalanine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His (Amber), proline This study P44S-His (Amber) 44 mutated to serine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His (Amber), alanine This study A46P-His (Amber) 46 mutated to proline, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His (Amber), proline This study P48T-His (Amber) 48 mutated to threonine, Cb^(R) pBAD/MBP-3C-RaxX- pBAD/MBP-3C-RaxX-His (Amber), arginine This study R55P-His (Amber) 55 mutated to proline, Cb^(R) pBAD/MBP-3C-RaxX-His 3C-RaxX-His coding for raxX from IXO685 This study (IXO685, Amber) cloned into pBAD/MBP-Myc-His A with a change of the Y41 codon from TAT to TAG for incorporation of the non-standard amino acid sulfotyrosine, Cb^(R) Cp^(R)—cephalexin resistance, Km^(R)—kanamycin resistance, Sp^(R)—spectinomycin resistance, Cb^(R)—carbenicillin resistance *Numbers indicate the sequence as annotated in the PXO99 genome, c before the number indicates the reverse complement.

TABLE 2 Bacterial strains used in this study. Strain Relevant characteristics Source Xoo strains PXO99 Philippine race 6 strain PXO99Az, Cp^(R) Hopkins C M et al., (1992) Mol Plant Microbe Interact 5(6): 451-459 PXO99ΔraxST^(Sp) PXO99ΔraxST::Sp, Sp^(R) This study PXO99Δ1.7^(Sp) PXO99Δ1,287,700-1,289,400::Sp, Cp^(R)Sp^(R) This study PXO99Δ1.0^(Sp) PXO99Δ1,282,000-1,283,000::Sp, Cp^(R)Sp^(R) This study PXO99ΔraxX PXO99ΔraxX(marker free), Cp^(R) This study PXO99ΔraxST PXO99ΔraxST(marker free), Cp^(R) This study IXO685 Field isolate of Xoo taken from diseased rice in Mishra D, et Kerala, India, Cp^(R) al. (2013) PLoS One 8(11): e81996 E. coli strains C321.ΔA.exp All UAG codons mutated to UAA and RF1 Lajoie M J, et removed al. (2013) Science 342(6156): 35 7-360 DH5α Used for general cloning EC100D Used for pJP5603-based cloning, pir⁺ BL21(DE3) F ompT gal dcm Ion hsdSB(rB− mB−) λ(DE3 [lacl lacUV5-T7 gene 1 ind1 sam7 nin5]), used for expressing raxST in pET28a(+) Cp^(R)—cephalexin resistance, Km^(R)—kanamycin resistance, Sp^(R)—spectinomycin resistance

TABLE 3 Marker genes and Primers used for qPCR. Gene Annotated function Primer name Primer Sequence (5′-3′) PR10b pathogenesis-related  PR10b-Q1 GTCGCGGTGTCGGTGGAGAG Bet v I family protein, PR10b-Q2 ACGGCGTCGATGAATCCGGC putative ribonuclease, induced by SA and  during fungal infection (McGee J D, Hamer J E, & Hodges T K (2001) Mol  Plant Microbe Interact 14(7):877-886) Os04g10100 Momilactone A synthase, Os04g10100-Q1 AAATGATTTGGGACCAGTCG required for phytoalexin Os04g10100-Q2 GATGGAATGTCCTCGCAAAC production, induced by elicitors and UV  radiation (Shimura K,  et al. (2007) J Biol  Chem 282(47):34013- 34018; Atawong A, Hasegawa M, & Kodama O (2002) Bioscience, biotechnology, and biochemistry 66(3):566- 570) Os12g36830 pathogenesis-related  Os12g36830-Q1 GAGAAGCTCATCCTCGACGG or RSOsPR10 Bet v I family protein, Os12g36830-Q2 GCATACTCGGTAGGGTGAGC induced by abiotic  stress, JA and fungal  infection (Hashimoto M, et al. (2004) Plant &  cell physiology  45(5):550-559) Os06g37224 ent-kaurene oxidase, Os06g37224-Q1 GAGAAGCTCATCCTCGACGG or OsKOL5 phytoalexin production, Os06g37224-Q2 GCATACTCGGTAGGGTGAGC induced by elicitors  (Itoh H, et al. (2004)  Plant molecular biology 54(4):533-547) Actin Actin-Q1 TCGGCTCTGAATGTACCTCCTA Actin-Q2 CACTTGAGTAAAGACTGTCACTTG

Example 12: Enhancement of Root Growth

This example shows, e.g., that sulfated raxX actively promotes root growth in rice, a model cereal crop and also a model for bioenergy feedstocks.

Sulfated RaxX induces root growth in rice. Both Kittake and TP309 rice seeds were germinated on MS agar media containing 100 nM sulfated RaxX21 (RaxX21-sY) or 100 nM nonsulfated RaxX21 (RaxX21-Y). RaxX21 is a 21 amino acid peptide of RaxX, which can activate XA21-mediated immunity on rice. Root length of the seedling was measured after 5 days. The RaxX21-sY treated rice seeds had significantly longer roots than Mock (water) treated or RaxX21-Y treated seeds (t-test, P<0.01 (n>35)).

Sulfated RaxX21 (RaxX21-sY) promotes growth of Arabidopsis thaliana seedling roots. Arabidopsis thaliana Col0 seedlings were sterilized with 30% bleach, washed 5 times with water, and placed in the dark at 4° C. for 4 days. The seeds were placed on plates containing ½ Murashige and Skoog media with vitamins (Caisson) plus 1% sucrose, pH 5.7, solidified with 0.5% Phytagel (Sigma). Plates were treated with water, 100 nM RaxX21-Y, or 100 nM RaxX21-sY. The plates were incubated vertically under continuous light for 6 days. RaxX21-sY (i.e., sulfated) had statistically longer root lengths after 6 days compared to RaxX21-Y (i.e., unsulfated) or mock treated controls at a statistical significance at α=0.05 based on the Tukey Kramer honestly significant difference test.

Example 13

We have recently shown that the rice receptor XA21 recognizes a sulfated protein, called RaxX, produced by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). This recognition triggers a robust and effective immune response (Song W Y et al., 1995, Science, 270(5243): 1804-1806; Pruitt R N et al., 2015, Science Advances). A synthetic 21-amino acid sulfated derivative of RaxX (RaxX21-sY) from Xoo strain PXO99 (FIG. 39A) is sufficient to activate XA21-mediated immune responses (Pruitt R N et al., 2015, Science Advances).

Sequence analysis revealed that RaxX21 is similar to the peptide hormone PSY (plant peptide containing sulfated tyrosine), which promotes cellular proliferation and expansion in Arabidopsis (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338) (Pruitt R N et al., 2015, Science Advances). Arabidopsis PSY1 (AtPSY1) is the best-characterized member of the plant PSY peptide family. AtPSY1 is an 18-amino acid glycopeptide with a single sulfotyrosine residue (FIG. 39A) (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338) that is secreted, processed and promotes root elongation primarily through regulation of cell size. AtPSY1 is widely expressed in Arabidopsis tissues (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338). AtPSY1 promotes acidification of the apoplastic space through activation of membrane proton pumps (Fuglsang A T et al., 2014, Plant J., 80(6): 951-964). This acidification is thought to activate pH-dependent expansins and cell wall remodeling enzymes that loosen the cellulose network (Cosgrove D J, 2000, Plant Physiol Biochem., 38(1-2): 109-124; Hager A., 2003, J Plant Res., 116(6): 483-505). Concomitant water uptake by the cell leads to cellular expansion. In addition to PSY, plants produce two other classes of tyrosine sulfated peptides: phytosulfokine (PSK) (Matsubayashi Y, Sakagami Y, 1996, Proc Natl Acad Sci USA, 93(15): 7623-7627) and root meristem growth factor (RGF) (Matsuzaki Y et al., 2010, Science, 329(5995): 1065-1067), PSK and RGF are also processed, secreted, and play a role in regulation of growth.

Here we demonstrate that RaxX peptides derived from diverse Xanthomonas species promote root growth, mimicking the growth promoting activities of PSY peptides. We also show that, unlike RaxX, PSY peptides do not activate XA21-mediated immunity. Thus, XA21 is a highly selective immune receptor capable of specifically recognizing the bacterial mimic. Based on these findings we propose a model whereby Xoo and other Xanthomonas strains produce RaxX to reprogram the host environment by hijacking PSY signaling. XA21 later evolved to recognize and respond to specifically to RaxX.

Materials and Methods

Identification of Putative RaxX Proteins

Putative PSY-like proteins were identified by NCBI Protein BLAST analysis using the default settings for short sequences (Altschul S F et al., 1990, J Mol Biol., 215(3): 403-410). For S. lycopersicum BLAST was performed using the Sol Genomics Network with the BLOSUM 62 algorithm (https://solgenomics.net/tools/blast/). Proteins were identified from a single source for each plant: A. thaliana Col-0 (refseq_protein, taxid: 3702), O. sativa Nipponbare (refseq_protein, taxid: 39947), Triticum aestivum Chinese Spring (taxid:4565), Musa acuminata subsp. Malaccensis (refseq_protein, taxid 214687), S. lycopersicum cv. Heinz 1706 (ITAG release 2.40). BLAST was initially performed with the 18 amino acid sequence of AtPSY1 (DYGDPSANPKHDPGVPPS). Criteria for selection were as follows: (1) Candidates must match the query with an expect-value ≤20 for NCBI Protein BLAST analysis (PAM 30 algorithm), (2) Candidates must have an invariant Asp-Tyr at the beginning of the query, (3) The full length protein must be between 60 and 200 amino acids with the PSY like motif in the second half, (4) The protein must be predicted to have a secretion signal by SignalP 4.0 (Petersen T N et al., 2011, Nat Methods, 8(10): 785-786). Additional candidates were identified by subsequent iterative BLAST with the 18 amino acid RaxX sequences from candidate RaxX proteins identified in the initial BLAST. The final list is shown in FIG. 45A and FIG. 45B. If multiple splicing variants were identified in the searching, only one is listed.

Sequence Analysis and Visualization

Sequence alignments in FIG. 39A was generated with Geneious software using default parameters (Kearse M et al., 2012, Bioinformatics, 28(12): 1647-1649). Sequence logos were constructed using WebLogo (Schneider T D, Stephens R M, 1990, Nucleic Acids Res., 18(20): 6097-6100; Crooks G E et al., 2004, Genome Res., 14(6): 1188-1190) with the 13-amino acid PSY-like sequences from the RaxX sequences shown in Table 51 and the PSY sequences in FIG. 45A and FIG. 45B. The bit score for a given residue indicates the conservation at that position, while the size of the individual letters within the stack indicate relative frequency of that amino acid at the position.

Arabidopsis Growth Conditions

All Arabidopsis thaliana used this study were in the Col-0 background. The AtTPST mutant, tpst-1, (SALK_009847) and homozygous Atlg72300 mutant (SALK_072802C) were obtained from the Arabidopsis Biological Resource Center (ARBC). A homozygous tpst-1 line was isolated from progeny of the SALK_009847 seeds. The AtPSKR1/AtPSKR2/Atlg72300 triple receptor mutant (Mosher S, Kemmerling B, 2013, Plant Signal Behav., 8(5): e24119) was obtained from Birgit Kemmerling's laboratory. Plants were grown on the indicated media or on Sungro professional growing mix under continuous light.

RaxX and PSY1 Peptides

The peptides used in this study are listed in Table S2. All peptides other than RaxX21-Y are tyrosine sulfated as indicated (Y^(S)). The synthetic AtPSY1 peptide used in these experiments lacks the hydroxy- and L-Ara₃-modifications at the C-terminus. The natural processed, modified state of OsPSY1a is not known. The 18-amino OsPSY1a acid peptide was synthesized based on alignment with AtPSY1. RaxX13-sY was obtained from Peptide 2.0. All other peptides were obtained from Pacific Immunology. The peptides were resuspended in ddH₂O.

Arabidopsis Root Growth Assays

Arabidopsis seeds were treated with 30% bleach for 12 minutes and then washed 4-5 times with autoclaved water. Sterilized seeds were incubated in the dark at 4° C. for 3-4 days. Plates were prepared with 0.5× Murashige and Skoog (MS) medium with vitamins (Caisson, MSP09), 1% sucrose, pH 5.7, 0.5% Phytagel (Sigma, P8169). Peptide (or water for mock treatments) was added to the indicated concentration (from a 1 mM stock) just before pouring into a plate. Seeds were placed on the plate (20 seeds per plate), and the lids were secured with Micropore surgical tape (1530-0). Plates were incubated vertically under continuous light (55 μmol m⁻² s⁻¹) at 24° C. Seedlings with delayed germination were marked after 3 days, and were not included in the analysis. Root lengths were measured after 8 days.

Arabidopsis Live Imaging of Root Growth

Live imaging of roots was performed as described previously with modifications to the media (Duan L et al., 2013, Plant Cell, 25(1): 324-341; Geng Y et al., 2013, Plant Cell, 25(6): 2132-2154). Sterilized tpst-1 seeds were grown on 1% agar media containing 1× MS nutrients (Caisson, MSP01), 1% sucrose, and 0.5 g l⁻¹ MES, adjusted to pH 5.7 with KOH. After 6 days, seeds were transferred to 0.5% Phytagel (Sigma, P8169) media containing 0.5× MS (Caisson, MSP09, 1% sucrose, and 0.5 g l⁻¹ MES, adjusted to pH 5.7 with KOH. Imaging and semiautomated image analysis were performed as described previously (Geng Y et al., 2013, Plant Cell, 25(6): 2132-2154).

Rice Root Growth Assays

Seeds of Oryza sativa sp. japonica cultivars Kitaake or Taipei 309 (TP309) were dehusked and sterilized with 30% bleach for 30 min. The seeds were washed 4-5 times with water and plated to cups with 50 mL 0.5× MS (Caisson MSP09), 1% sucrose (pH 5.7 with KOH/NaOH) containing 0.25% Phytagel. Peptides were added to 100 nM just before pouring into the cups. 20 seedlings were added per cup. The seedling roots were measured after 4-6 day incubation in a 28° C. chamber with 13 h/11 h light/dark cycle and a light intensity of 15 μmol m⁻² s⁻¹.

ROS Assays

Kitaake and a transgenic derivative expressing XA21 under the ubiquitin promoter (XA21-Kitaake) were greenhouse grown. ROS assays were carried out using leaves of 6-week-old rice plants as described previously (Pruitt R N et al., 2015, Science Advances). Briefly, leaves of were cut longitudinally along the mid vein and then transversely into 1- to 1.5-mm-thick leaf pieces. After overnight incubation on sterile water, leaf pieces were transferred into a 96-well white plate (2 pieces per well). Each well contained 100 μl of excitation solution [0.2 mM L-012 (Wako) and 50 μg ml⁻¹ horseradish peroxidase (Sigma)]. The indicated concentration of peptides was added (or water for mock control), and chemiluminescence was measured for 90 minutes with a TriStar (Berthold) plate reader.

Supplemental Information

Table S1. RaxX13 Sequences from Diverse Xanthomonas Sources.

The RaxX sequences were grouped into three categories X. oryzae (blue), X. translucens (green) and other Xanthomonas strains (orange). The full length sequences of the strains are shown in FIG. 46. A short-hand letter/number code was given to each RaxX13 sequence. RaxX proteins that are identical over the 13 amino acid PSY-like region have the same letter. Proteins that are identical over the entire RaxX sequence have the same letter and number.

TABLE S1 RaxX13 sequences from diverse Xanthomonas sources. # RaxX13 Species Pathovar Strain Host PMID/BioSam A1 DYPPPGANPKHDP X. oryzae pv. oryzae PXO99A Rice 1353345 X. oryzae pv. oryzae KACC10331 Rice X. oryzae pv. oryzae PXO83 Rice 1353345 A2 DYPPPGANPKHDP X. oryzae pv. oryzae PXO86 Rice 1353345 X. oryzae pv. oryzae MAFF311018 Rice X. oryzae — ATCC35933 † Rice B1 DYPPSGPNPKHDP X. oryzae pv. oryzae NAI8 Rice 21515727 B2 DYPPSGPNPKHDP X. oryzae pv. oryzae AXO1947 Rice B3 DYPPSGPNPKHDP X. oryzae — X8-1A Rice 21515727 C1 DYPPAGPSTKHDP X. oryzae — X11-5A Rice 21515727 D1 DYPPSGPNTKHDP X. oryzae pv. oryzicola BLS279 Rice 26257749 X. oryzae pv. oryzicola RS115 Rice 26257749 X. oryzae pv. oryzicola BXOR1 Rice 26257749 X. oryzae pv. oryzicola MAI10 Rice 26257749 X. oryzae pv. oryzicola L8 Rice 26257749 X. oryzae pv. oryzicola B8-12 Rice 26257749 X. oryzae pv. oryzicola CFBP 7331 Rice X. oryzae pv. oryzicola CFBP 7342 Rice X. oryzae pv. oryzicola CIX4 Rice X. oryzae pv. oryzicola YM15 Rice D2 DYPPSGPNTKHDP X. oryzae pv. oryzicola BLS256 Rice 26257749 X. oryzae pv. oryzicola CFBP 2286 Rice 26257749 D3 DYPPSGPNTKHDP X. oryzae pv. oryzicola BAI5=CFBP  Rice 26257749 7341 D4 X. campestris pv. leersiae NCPPB 4346 Southern  21515727 Cutgrass X. oryzae IXoo651 Rice D5 X. oryzae IXoo685 Rice X. oryzae IXoo1221 Rice E1 DYPPPGANTKHDP X. euvesicatoria 85-10 85-10 Pepper 16237009 X. axonopodis pv. dieffenbachiae LMG 695 Anthurium SAMN0290399 X. alfalfae sb. citrumelonis F1 Citrus 21908674 X. perforans 91-118 Pepper &  26089818 Tomato X. axonopodis pv. phaseoli XCP123 Lima Bean 26500625 X. axonopodis pv. manihotis CIO151 Cassava 22699502 E2 DYPPPGANTKHDP X. axonopodis pv. manihotis LMG 784 Cassava 25085494 X. axonopodis pv. manihotis UG28 Cassava 22699502 X. axonopodis pv. manihotis Xam672 25085494 X. axonopodis pv. manihotis UG39 Cassava 22699502 X. axonopodis pv. manihotis UA536 Cassava 22699502 F1 DYPPPGANDRHNP X. maliensis — M97 † Rice 25588569 G1 DYAPPGSNDRHDP X. campestris pv. musacearum NCPPB 2005 Ensete 24704974 ventricosum X. campestris pv. musacearum NCPPB 4379 Banana 24704974 X. campestris pv. musacearum NCPPB 4380 Banana 24704974 X. campestris pv. musacearum NCPPB 4381 Banana 24704974 X. campestris pv. musacearum NCPPB 4384 Banana 24704974 X. campestris pv. musacearum NCPPB 4392 Banana 24704974 X. campestris pv. musacearum NCPPB 4394 Banana 24704974 G2 DYAPPGSNDRHDP X. vasicola pv. vasculorum NCPPB 702 Sugarcane 24704974 X. vasicola pv. vasculorum NCPPB 890 X. vasicola pv. vasculorum NCPPB 895 X. vasicola pv. vasculorum NCPPB 1326 Sugarcane 24704974 X. vasicola pv. vasculorum NCPPB 1381 Sugarcane 24704974 H1 DYPPPGSNNRHDP X. vasicola pv. vasculorum NCPPB 206 Maize 24704974 X. vasicola pv. vasculorum NCPPB 2649 I1 DYPPTGPNTRHDP X. axonopodis pv. vasculorum NCPPB 900 Sugarcane 25263632 J1 DYPPSAANDRHDP X. axonopodis pv. axonopodis DSM 3585 † Imperial SAMN02646552 Grass K1 DYPPPSSNGRHDP X. translucens pv. undulosa Xtu 4699 Wheat & 26729225 Barley X. translucens — CR31 Wheat & 26729225 Barley X. translucens — CS2 Wheat & 26729225 Barley X. translucens — CS22 Wheat & 26729225 Barley X. translucens — CS4 Wheat & 26729225 Barley X. translucens — DAR61454 Wheat & 24416331 Barley X. translucens — LB10 Wheat & 26729225 Barley X. translucens — LB5 Wheat & 26729225 Barley X. translucens — LG48 Wheat & 26729225 Barley X. translucens — LG54 Wheat & 26729225 Barley X. translucens — LW16 Wheat & 26729225 Barley X. translucens — P3 Wheat & 26729225 Barley X. translucens — XT-Rocky Wheat & 26729225 Barley X. translucens — XT130 Wheat & 26729225 Barley X. translucens — XT5523† Wheat & 26729225 Barley X. translucens — XT5770 Wheat & 26729225 Barley L1 DYAPPSSNGRHDP X. translucens — XT5791 Wheat & 26729225 Barley M1 DYPPPHSNDRHDP X. translucens pv. translucens DSM 18974† Barley — X. translucens pv. graminis CFBP 2053 — 26450740 X. translucens pv. graminis ART-Xtg29 Forage grass 23578314 X. translucens pv. translucens UPB787 — — X. translucens — XT8 Barley 26729225 N1 DYPPTGANDKHDP X. translucens pv. poae LMG 728 Poa trivialis SAMEA3486272 O1 DYPPSGPNTRHDP X. translucens pv. arrhenatheri LMG 727 Arrhenatherum  SAMEA3486180 elatius P1 DYPTAGANRKHDP X. translucens pv. phlei LMG 730 Phleum  SAMEA3486273 pratense Q1 DYPTAGANRKHDP X. translucens pv. cerealis CFBP 2541† Wheat & 25676771 Barley X. translucens — XT123 Wheat & 26729225 Barley

TABLE S2 Synthetic peptides used in this study Peptide Sequence Sequence Source RaxX21-Y HVGGGDYPPPGANP Xoo PXO99 KHDPPPR RaxX21-sY HVGGGDY^(S)PPPGAN Xoo PXO99 PKHDPPPR* RaxX16-sY DY^(S)PPPGANPKHDP Xoo PXO99 PPR RaxX13-sY DY^(S)PPPGANPKHDP Xoo PXO99 AtPSY1 DY^(S)GDPSANPKHDP A. thaliana Col-0 GVPPS OsPSY1a DY^(S)PAPGANPRHNP O. sativa Nipponbare KRPPG PSK Y^(S)IY^(S)TQ A. thaliana Col-0 RaxX24- LWKHVGGGDY^(S)PPS X. oryzae pv.  Xoc-sY GPNTKHDPPPP oryzicola BSL256 RaxX24- LWKHVGGGDY^(S)PPP X. euvesicatoria 85-10 Xe-sY GANTKHDPPPK RaxX24- LWKHVGGGDY^(S)APP X. campestris pv.  Xcm-sY GSNDRHDPPKP musacearum NCPPB4394 *Y^(S) indicates sulfated tyrosine. Results RaxX is Similar in Sequence to PSY Peptides

The region of similarity between RaxX from Xoo and AtPSY1 corresponds to amino acids 40-52 of RaxX. RaxX and AtPSY1 share 10 identical residues over this region (FIG. 39A). RaxX is sulfated by the bacterial sulfotransferase RaxST on Y41, which corresponds to the sulfated residue of AtPSY1 (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338; Pruitt R N et al., 2015, Science Advances). An aspartate precedes the sulfated tyrosine in both RaxX and AtPSY1. The presence of a nearby acidic residue is a common hallmark of tyrosine sulfation sites (Moore K L, 2009, Proc Natl Acad Sci USA, 106(35): 14741-14742).

We extended our analysis to include PSY and RaxX peptides from diverse species (FIGS. 45A, 45B and 46, Table S1). BLAST search using the 18 amino acid AtPSY1 as a query identified 8 PSY-like proteins in rice (FIG. 45A and FIG. 45B). One of the rice PSY proteins, OsPSY1 (Os05g40850), has four nearly identical PSY-like repeats, the first of which (OsPSY1a) is shown in FIG. 1. Analysis of Arabidopsis using the same criteria also revealed a total of eight PSY like proteins including the three that had been previously identified (FIG. 45A and FIG. 45B) (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338; Matsubayashi Y., 2014, Annu Rev Plant Biol., 65: 385-413). We also identified PSY-like proteins in tomato, banana and wheat, three diverse and economically important crops (FIG. 45A and FIG. 45B). Alignment of PSY peptides from these different species revealed a highly conserved 13-amino acid region beginning with the aspartate-tyrosine residue pair (FIG. 45A and FIG. 45B). This 13-amino acid sequence corresponds precisely to the region of sequence similarity between RaxX and AtPSY1 (FIG. 39A).

Alignment of the RaxX sequences from diverse strains reveals a region of high conservation immediately around the tyrosine, which is sulfated in Xoo strain PXO99 (FIG. 46). Sequence logos were constructed for the PSY-like motif using the identified RaxX and PSY sequences (FIG. 39B). These logos further highlight the similarity of 13-amino acid region of RaxX and PSY sequences. Residues that are highly variable in RaxX are also highly variable in PSY. Based on the similarity of RaxX and PSY peptides and the finding that RaxX is also tyrosine sulfated (Pruitt R N et al., 2015, Science Advances), we hypothesized that RaxX serves as a functional mimic of PSY peptides and that RaxX may have PSY-like activity.

RaxX Promotes Root Growth Similar to PSY Peptides

To test our hypothesis that RaxX can mimic the growth stimulating activity of PSY-like peptides, we evaluated the effect of RaxX21 treatment on root growth of Arabidopsis seedlings. RaxX21-sY promoted root growth in a similar manner to that observed for AtPSY1 in Arabidopsis (FIGS. 40A and 40B). After 8 days on media containing 100 nM RaxX21-sY, the average root length of Col-0 seedlings was 61 mm whereas seedlings grown on plates without peptide had an average root length of 54 mm. Similar root growth-promoting effects were observed in experiments using AtPSY1 and OsPSYla peptides (FIGS. 40A and 40B).

We also performed root growth experiments on an Arabidopsis line lacking AtTPST, the tyrosine sulfotransferase responsible for modification of PSY, PSK and RGF peptides (Komori R et al., 2009, Proc Natl Acad Sci USA, 106(35): 15067-15072; Matsuzaki Y et al., 2010, Science, 329(5995): 1065-1067). tpst-1 mutant plants are dwarf and have stunted roots (Komori R et al., 2009, Proc Natl Acad Sci USA, 106(35): 15067-15072). Because this mutant lacks endogenous PSY, PSK and RGF signaling, effects of exogenous application of sulfated peptides can be better quantified (Igarashi D, Tsuda K, Katagiri F, 2012, Plant J., 71(2): 194-204; Mosher S, Kemmerling B, 2013, Plant Signal Behay., 8(5): e24119). Consistent with earlier reports, we observed that mock treated tpst-1 mutant seedlings have much shorter roots than Col-0 (FIGS. 40C and 40D). Treatment of tpst-1 plants with RaxX21-sY or AtPSY1 increases root growth 1.5-2 fold relative to mock treatment (FIGS. 40C and 40D).

We determined the minimum concentration of RaxX21-sY needed to induce root growth in Arabidopsis. tpst-1 seeds were grown on plates containing 0.1-250 nM peptide. RaxX21-sY was effective at inducing root growth at concentrations in the low nanomolar range (FIG. 47). This activity is comparable to PSK (FIG. 47). Nonsulfated RaxX21 (RaxX21-Y) also promoted root growth, but was less active than the sulfated version (FIGS. 40A to 40D, 47). AtPSY1 was less active than RaxX21-sY and PSK. We hypothesize that the reduced potency of the synthetic AtPSY1 used in this study was due to the lack of glycosylation (See materials and methods). Glycosylation of AtPSY1 was previously shown to be important for full activity (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338).

We next used a live root imaging system (Duan L et al., 2013, Plant Cell, 25(1): 324-341; Geng Y et al., 2013, Plant Cell, 25(6): 2132-2154) to assess changes in root growth rate upon exposure to RaxX21-sY. Root growth of tpst-1 seedlings on plates containing 250 nM RaxX21-sY, AtPSY1 or no peptide (Mock) was monitored over 24 h. Within 4-5 hours, seedlings grown on RaxX21-sY- or AtPSY1-containing plates had an increased root growth rate compared to seedlings on mock plates (FIG. 40E).

To determine if RaxX induces root growth using the same signaling pathway as AtPSY1, we grew Arabidopsis seedlings on plates containing both RaxX and AtPSY1 peptides. Roots of Arabidopsis seedlings grown on plates containing 100 nM RaxX21-sY and 100 nM AtPSY1 were similar in length to those grown on plates with 100 nM RaxX21-sY alone (FIG. 48). Similar results were observed when seedlings were co-treated with 100 nM RaxX21-sY and 100 nM PSK (FIG. 48). The observation that RaxX, AtPSY1, and PSK do not have additive effects on root growth suggests that these peptides induce root growth via the same pathway. Alternatively it may be that the 100 nM RaxX21-sY treatment already reached the maximum growth potential (Matsuzaki Y et al., 2010, Science, 329(5995): 1065-1067).

Because RaxX21-sY comes from the rice pathogen Xoo, we tested whether this peptide also has growth promoting activity in rice seedlings. AtPSY1 and RaxX21-sY treatment significantly enhanced root growth on rice varieties Tapei 309 (FIG. 40F) and Kitaake (FIG. 49). Collectively, these results indicate that RaxX21-sY promotes root growth in a similar manner to PSY and PSK peptides in both Arabidopsis and rice.

Atlg72300 is not Required for Induction of Root Growth by RaxX or AtPSY1

The leucine-rich repeat receptor kinase encoded by Atlg72300 has been proposed to serve as the AtPSY1 receptor (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338). We therefore tested if Atlg72300 is required for perception of RaxX21-sY. For these assays we used the Atlg72300 mutant line SALK_072802C. This is the same line used in all published studies of PSY1/Atlg72300, (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338; Mosher S, Kemmerling B, 2013, Plant Signal Behay., 8(5): e24119; Mosher S et al., 2013, Plant J, 73(3): 469-482; Fuglsang A T et al., 2014, Plant J., 80(6): 951-964; Mahmood K et al., 2014, BMC Genomics, 15: 441), and was shown to have the lowest transcript level of available mutants (Fuglsang A T et al., 2014, Plant J., 80(6): 951-964). We independently validated the mutant genotype (FIG. 50). We found that treatment of the Atlg72300 mutant line with either RaxX21-sY or AtPSY1 increased root growth in a similar manner to that observed for treatment of wild type Col-0 seedlings (FIGS. 40A, 40B, and 41A). We also found that a mutant lacking Atlg72300 and the homologous PSK receptors, AtPSKR1 and AtPSKR2, also responds to RaxX and AtPSY1 treatment (FIG. 41B). These results indicate that Atlg72300 is not required for perception of RaxX21-sY or AtPSY1.

RaxX21-sY and PSY do not Attenuate Elf18-Induced Growth Inhibition

Exogenous addition of PSK has previously been shown to attenuate the Arabidopsis immune response to biotrophic pathogens (Igarashi D, Tsuda K, Katagiri F, 2012, Plant J., 71(2): 194-204; Mosher S, Kemmerling B, 2013, Plant Signal Behay., 8(5): e24119; Mosher S et al., 2013, Plant J, 73(3): 469-482). Although PSK and AtPSY1 share no sequence similarity, they have nevertheless been hypothesized to serve similar roles (Mosher S, Kemmerling B, 2013, Plant Signal Behay., 8(5): e24119; Mosher S et al., 2013, Plant J, 73(3): 469-482); Matsubayashi Y., 2014, Annu Rev Plant Biol., 65: 385-413). Thus, we hypothesized that induction of PSY signaling by PSY or RaxX21-sY may also attenuate plant immune responses. To test this hypothesis, we employed a seedling growth inhibition assay. Arabidopsis seedlings were grown in the presence of the bacterial elicitor elf18, which causes activation of immune response and impairs growth. We demonstrated that co-incubation of seedlings with PSK attenuates elf18-mediated growth inhibition as previously reported (Igarashi D, Tsuda K, Katagiri F, 2012, Plant J., 71(2): 194-204) (FIG. 51). However, RaxX21-sY and AtPSY1 do not prevent elf18-triggered growth inhibition in Arabidopsis under the conditions tested (FIG. 51). These results indicate that RaxX21-sY and PSY1 do not have the same effects on immune modulation as PSK in Arabidopsis seedlings in response to elf18 treatment.

RaxX and PSY Peptides Differentially Activate PSY-Like Growth Promotion and XA21-Immune Responses

Activation of XA21-mediated immunity by RaxX21-sY triggers a number of immune responses including production of reactive oxygen species (ROS), induction of marker gene expression, and production of ethylene (Pruitt R N et al., 2015, Science Advances). These immune responses are tightly regulated, because aberrant activation of immunity can have negative effects on plant growth and health (Spoel S H, Dong X, 2012, Nat Rev Immunol., 12(2): 89-100; Rodriguez E et al., 2015, FEBS J.). We therefore hypothesized XA21 would specifically recognize RaxX but not the homologous PSY peptides, because unregulated recognition of endogenous PSY1 would have deleterious effects.

We have previously shown that RaxX21-sY treatment induces robust ROS production in rice leaves expressing XA21 (Pruitt R N et al., 2015, Science Advances). Therefore, to assess XA21-mediated recognition of the sulfated peptides, we measured ROS production XA21 rice leaves upon treatment with water, RaxX21-sY, AtPSY1, or OsPSY1a (FIG. 42A). Unlike RaxX21-sY, AtPSY1 and OsPSY1a failed to induce ROS production in XA21 rice leaves. Robust ROS production was not observed in rice leaves lacking XA21 (FIG. 42B). PSK also failed to activate XA21-mediated immune response (FIGS. 42A and 42B). These results suggest that the XA21 and PSY receptor(s) have different specificities. PSY signaling is activated by both PSY and RaxX (FIGS. 40A to 40F), whereas the XA21-mediated immune response is only activated by RaxX.

To further delineate the region of RaxX required for PSY-like activity and activation of XA21, we synthesized two smaller RaxX peptides based on similarity to AtPSY1. RaxX16-sY begins with the aspartate (D40) at the beginning of the PSY-like motif (FIG. 39A). RaxX13-sY also begins with D40 but is C-terminally truncated relative to RaxX21-sY and RaxX16-sY (FIG. 39A). RaxX13-sY contains the region of highest similarity shared between the RaxX and PSY peptides (FIGS. 39A, 39B, 45A and 45B). Both the RaxX13-sY and RaxX16-sY peptides are still capable of promoting root growth in Arabidopsis and rice (FIGS. 42C and 42D). We next tested whether these peptides could activate XA21-mediated immunity in the same manner as RaxX21-sY (Pruitt R N et al., 2015, Science Advances). For this purpose, ROS production was measured in detached XA21 rice leaves treated with water, RaxX13-sY, RaxX16-sY, or RaxX21-sY. RaxX16-sY and RaxX21-sY triggered a ROS response characteristic of the XA21-mediated immune response. In contrast, treatment with RaxX13-sY did not induce ROS production in XA21 rice leaves (FIG. 42A). Thus, RaxX13-sY is able to induce AtPSY1 like growth effects, but fails to activate an XA21-mediated immune response. These experiments reveal that RaxX residues 53-55, which are present in RaxX16 but not RaxX13, are important for activation of XA21 but are not required for root growth promoting activity.

RaxX from Diverse Xanthomonas Species have PSY Activity

We next asked whether RaxX from other Xanthomonas strains also have PSY-like activity. To address this question, we synthesized 24-amino acid peptides covering the PSY-like region for three different RaxX sequences from X. oryzae pv. oryzicola strain BSL256 (RaxX24-Xoc-sY), X. campestris pv musacearum strain NCPPB4394 (RaxX24-Xcm-sY), and X. euvesicatoria strain 85-10 (RaxX24-Xe-sY) (Table S2). Xoc, Xcm, and Xe are pathogens of rice, banana, and tomato/pepper, respectively (Table S1). Xoc colonizes the mesophyll of rice, whereas Xoo colonizes the xylem. All three RaxX sulfated peptides promoted root growth on Arabidopsis seedlings in a manner similar to that of RaxX21-sY derived from Xoo strain PXO99 (FIG. 43). In other words, the proteins encoded by diverse allelic variants of raxX retain PSY like activity. These results demonstrate that the use of RaxX as a mimic of plant PSYs is employed by many Xanthomonas species that infect diverse plant species.

Discussion

In a classical evolutionary arms race, both the pathogen and host develop and deploy an arsenal of strategies to infect or resist their partner. For example, many pathogens secrete an array of molecular factors designed to manipulate host biology and suppress the immune response. In turn, plants have developed a set of immune receptors that recognize these molecules or their activities and launch mechanisms to destroy the pathogen, which the pathogen then tries to counter.

The findings reported here and in previous studies, suggest a model where Xoo produces, sulfates, and secretes a peptide that mimics PSY peptides (da Silva F G et al., 2004, Mol Plant Microbe Interact, 17(6): 593-601; Pruitt R N et al., 2015, Science Advances) (FIG. 44). Plants evolved the receptor XA21 to specifically recognize the bacterial mimic, allowing it to launch a defense response in the presence of the pathogen but not in the presence of the highly conserved PSY peptide hormones, which are predicted to be necessary for normal growth and development.

The hypothesis that RaxX is a mimic of PSY is well supported by the high level of sequence similarity (FIGS. 39A and 39B), the tyrosine sulfation status of RaxX and PSY peptides (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338; Pruitt R N et al., 2015, Science Advances), and the similar growth promoting activities of both peptides (FIGS. 40A to 40F, and 47 to 49). Significantly, both RaxX and PSY1 require a tyrosine sulfation for full activity. Tyrosine sulfation is an important posttranslational modification that mediates protein-protein interactions. Plants and animals employ tyrosine-sulfated proteins, to regulate growth, development, immunity and other biological processes. Tyrosine sulfated proteins in animal cells have roles in coagulation, leukocyte adhesion, HIV entry, and chemokine signaling (Farzan M et al., 1999, Cell, 96(5): 667-676; Moore K L, 2009, Proc Natl Acad Sci USA, 106(35): 14741-14742; Stone M J et al., 2009, N Biotechnol., 25(5): 299-317).

Due to the similar sequence and functional mimicry in root growth promotion we hypothesize that PSY1 and RaxX target a common cognate plant receptor. The leucine-rich repeat receptor kinase Atlg72300 was originally hypothesized to serve as the receptor for AtPSY1 based on the observation that the root length was not increased by endogenous AtPSY1 treatment in an Atlg72300 mutant (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338). However, the Atlg72300 mutant line still partially responds to AtPSY1 treatment in proton efflux experiments (Fuglsang A T et al., 2014, Plant J., 80(6): 951-964), and transcriptomics analysis reveals that many AtPSY1-regulated genes are regulated independently of Atlg72300 (Mahmood K et al., 2014, BMC Genomics, 15: 441). We found that RaxX and AtPSY1 still promote root growth in the absence of Atlg72300. Collectively, these findings indicate that Atlg72300 is not the receptor for PSY peptides or that it is not the only receptor. Additional work is required to understand how PSY and RaxX are perceived in plants.

The precise role of RaxX in Xoo biology is not known. Because bacteria have been demonstrated to employ bio-mimics to hijack the plants' endogenous systems and reprogram the host environment to facilitate pathogen infection (Weiler E W et al., 1994, FEBS Lett., 345(1): 9-13; Melotto M et al., 2006, Cell, 126(5): 969-980; Mitchum M G et al., 2012, Annu Rev Phytopathol, 50: 175-195; Chen S et al., 2015, Physiol., 167(1): 262-272), we hypothesize that Xoo may use RaxX in a similar manner. Xoo is a biotrophic pathogen and thus requires living host tissues, which ensures prolonged supply of the carbon and other nutrients necessary for bacterial survival. The ability of Xoo to promote the host growth would thus benefit a biotroph (Nino-Liu D O, Ronald P C, Bogdanove A J, 2006, Mol Plant Pathol., 7(5): 303-324; Fatima U, Senthil-Kumar M, 2015, Front Plant Sci., 6: 750).

Xanthomonads enters through hydathodes, natural openings in the leaf, or wounds and multiplies in the xylem or mesophyll tissues. To date, growth promoting activities for RaxX or PSY1 have only been demonstrated on roots. We used induction of root growth as an indicator of PSY-like activity in this study because this is a robust well-characterized effect of AtPSY1. It is known, however, that AtPSY1 is widely expressed in various plant tissues (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338). Arabidopsis seedlings overexpressing AtPSY1 not only have longer roots, but also larger cotyledons (Amano Y et al., 2007, Proc Natl Acad Sci USA, 104(46): 18333-18338). Recently, a PSY-like peptide in soybean was shown to be translocated from the roots to the xylem (Okamoto S et al., 2015, Plant J., 84(3): 611-620). These findings suggest that PSY peptides likely have important unidentified roles outside of the roots.

The growth promoting properties of RaxX are reminiscent of the hypertrophy in tomato and pepper leaves induced by the Xe effector AvrBs3. AvrBS3 enhances transcription of host genes including auxin-induced and expansin-like genes that contribute to host cell enlargement (Marois E et al., 2002, Mol Plant Microbe Interact, 15(7): 637-646). This phenotype is thought to facilitate dissemination because the bacteria are able to multiply in the enlarged cells and escape from the infected site to other plants (Marois E et al., 2002, Mol Plant Microbe Interact, 15(7): 637-646; Kay S et al., 2007, Science, 318(5850): 648-651). Although we have not observed an effect of RaxX on disease lesion development on rice leaves (da Silva F G et al., 2004, Mol Plant Microbe Interact, 17(6): 593-601; Pruitt R N et al., 2015, Science Advances), the AvrBs3 example suggests a possible role for RaxX in bacterial maintenance, persistence or transmission.

In this paper we demonstrate that XA21 is able to recognize RaxX16, but not RaxX13 indicating that the C-terminal end of the RaxX16 sequence (RaxX amino acids 53-55) is required for XA21 recognition. Residues within the RaxX13 region are also important for recognition by XA21. In a previous study, we identified three residues (44, 46, and 48) of RaxX from Xoo that are involved in XA21 activation (Pruitt R N et al., 2015, Science Advances). Mutation of RaxX P44 and P48 completely abolished the immunogenic activity of RaxX on XA21-rice. Mutation of A46 had a partial effect. Interestingly, these residues are not required for root growth promoting activity. For example, RaxX24-Xoc contains amino acid differences at positions 44, 46 and 48, but is still capable of inducing root growth in Arabidopsis (FIGS. 43 and 46, Table 1).

Comparison of the RaxX-Xoo and RaxX-Xoc sequences with rice PSY sequences suggests the possibility that RaxX from the Xanthomonas strains have evolved to mimic different PSY peptides. The three residues from RaxX-Xoo (strain PXO99) which are required for recognition by XA21 are identical to those in OsPSY1a. In contrast, the amino acids of RaxX-Xoc (strain BSL256) are similar to those in OsPSY2. If these two peptides have evolved to mimic different PSY peptides, it would indicate that there are multiple PSY receptors in rice, which differentially recognize diverse PSY peptides. Multiple receptors have been reported for RGF peptides. It is not yet clear if the RGF receptors have different affinities for specific RGF peptides (Shinohara H et al., 2016, Proc Natl Acad Sci USA). Using multiple receptors and multiple ligands with different affinities would allow for a more complex and tunable signaling network.

To further investigate the possibility that RaxX may have evolved to mimic specific host PSY peptides, we compared the sequences of RaxX13 and PSY from various species (FIG. 39B). We did not observe a correlation between the sequences of RaxX from the pathogen and PSYs from a compatible host. However, alignment of the 13-amnio acid region did highlight variation at positions 5, 7, and 9. These residues correspond to RaxX amino acids 44, 46, and 48, which are important for XA21 recognition. Notably, the variation is not random. For example, the most common amino acids in position 5 of the sequences analyzed are serine and proline in both RaxX and PSY (FIG. 39B). The amino acids in this position could affect the ability of the peptides to activate specific PSY receptor(s), as they do for XA21. Alternatively, the PSY receptor(s) may simply be able to accommodate serine or proline at this position. Further research, including the characterization of the PSY receptor(s), will help address questions of specificity and lead to a greater understanding of PSY signaling.

The study of microbial mimicry of host molecules provides insight into both host and pathogen biology, and can lead to novel strategies for disease prevention (Gardner M R et al., 2015, Nature, 519(7541): 87-91). Recent studies of the JA receptor have provided new insight into selective recognition of endogenous hormones. The endogenous JA receptor is sensitive to both JA-Ile and the mimic coronatine. By making a structure-guided point mutation of a single amino acid, Zhang, et al generated a modified JA receptor which has strongly reduced sensitivity to coronatine while retaining endogenous JA-Ile recognition (Zhang L et al., 2015, Proc Natl Acad Sci USA, 112(46): 14354-14359. Arabidopsis with the modified JA receptor displayed enhanced resistance to coronatine producing Pseudomonas strains, and have a normal phenotype in the absence of infection (Zhang L et al., 2015, Proc Natl Acad Sci USA, 112(46): 14354-14359). The Zhang et al. study demonstrates how understanding of bacterial mimicry of host factors can be used to engineer plants with enhanced resistance to bacterial pathogens. The findings presented in this work provide another striking example of co-evolution between the host and pathogen and provide a framework for future work directed at understanding how XA21 and the PSY receptor(s) differentially recognize RaxX and endogenous PSY peptides.

In the claims appended hereto, the term “comprise” and variations thereof such as “comprises” and “comprising,” when preceding the recitation of a step or an element, are intended to mean that the addition of further steps or elements is optional and not excluded. All patents, patent applications, and other published reference materials cited in this specification are hereby incorporated herein by reference in their entirety.

Exemplary Sequences Discussed Herein

Exemplary Xoo raxX polypeptide:  (SEQ ID NO: 1) LWKHVGGGDYPPPGANPKHDPPPR. Exemplary Xoo raxX polypeptide: (SEQ ID NO: 2) PLDQRLWKHVGGGDYPPPGANPKHDPPPRNPGHH Exemplary Xoo raxX polypeptide: (SEQ ID NO: 3) KGRPEPLDQRLWKHVGGGDYPPPGANPKHDPPPRNPGHH. Exemplary Xoo raxX polypeptide: (SEQ ID NO: 4) HVGGGDYPPPGANPK. Consensus raxX polypeptide: (SEQ ID NO: 22) MNLSKKSPTKGAASLQRPAGAKGRPEPLDQRLWKHVGGGDYPPPGXNTKH DPPPNNGHH.

Nonredundant raxX alleles. raxX is present in other species (e.g., Xanthomonas perforans, Xanthomonas axonopodis manihotis, Xanthomonas axonopodis citrumelo) that are not included on this list because they are identical in sequence to one of those listed below.

>RaxX_Xac_F1 Xanthomonas axonopodis pv.  citrumelo F1 chromosome, complete genome  SEQ ID NO: 5 ATGAATCTGTCGAAAAAATCGCCAACCAATGGTGCGCAAGCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCAGCTGGAGCCCCTGGACCAGCGGCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCCAATACCAAGCAC GATCCGCCGCCCAAGAACGGGCACCACTGA >RaxX_Xam_UA303 Xanthomonas axonopodis pv.  manihotis str. UA303 UA303_contig73, whole genome  shotgun sequence >gi|390589119|gb|AKEM01000102.1| Xanthomonas axonopodis pv. manihotis str. UA303  UA303_contig73, whole genome shotgun sequence  SEQ ID NO: 6 ATGAACCTGTCGAAAAAATCGCCAACCAATGGTGCGCAAGCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCAGCTGGAGCCCCTGGACCAGCGTATGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCCAATACCAAGCAC GATCCGCCGCCCAAGAACGGGCACCACTGA >RaxX_Xcm_NCPPB4394 A new nucleotide sequence  entered manually  SEQ ID NO: 7 ATGAAACTATTGAAAAAATCGCCATCCAATGGCGCGGGATCGCTCCAGCG GTCGGCCGGCGCCAAGGGCCGGCTAGAGCCGCTGGACCAGCGTCTGTGGA AGCATGTCGGGGGTGGGGACTATGCCCCGCCGGGCTCGAATGACCGCCAC GACCCACCAAAGCCGCCGCCCAAAAAAGGGAACCCTTGA >RaxX_Xcv_NCPPB702 A new nucleotide sequence  entered manually  SEQ ID NO: 8 ATGGAACTATTGAAAAAATCGCCATCCAATGGCGTGGGATCGCTCCAGCG GTCGGCCGGCGCCAAGGGCCGGCTAGAGCCGCTGGACCAGCGTCTGTGGA AGCATGTCGGGGGTGGGGACTATGCCCCGCCGGGCTCGAATGACCGCCAC GACCCACCAAAGCCGCCGCCCAAAAAAGGGAACCCTTGA >RaxX_Xe_85-10 A new nucleotide sequence  entered manually  SEQ ID NO: 9 ATGAATCTGTCGAAAAAATCGCCAACCAATGGTGCGCAAGCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCAGCTGGAGCCCCTGGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCCAATACCAAGCAC GATCCGCCGCCCAAGAACGGGCACCACTGA >RaxX_Xo_ATCC35933 Xanthomonas oryzae ATCC 35933  K396DRAFT_scaffold00007.7_C, whole genome shotgun  sequence  SEQ ID NO: 10 ATGAACCACTCGAAAAAATCGCCCACCAAGGGTGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTCGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCGAATCCCAAGCAC GATCCGCCGCCGCGCAACCCCGGGCACCATTGA >RaxX_Xo_X8-1A Xanthomonas oryzae X8-1A  Xo_X8-1A_contig_259, whole genome shotgun sequence >gi|332357675|gb|AFHL01000259.1| Xanthomonas  oryzae X8-1A Xo_X8-1A_contig_259, whole genome  shotgun sequence  SEQ ID NO: 11 ATGAACCTGTCGAAAAAATCGCCAACCAAGGGTGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTGGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGTCGGGCCCGAATCCCAAGCAC GATCCACCGGGCAACAACGGGCACCATTGA >RaxX_Xo_X11-5A Xanthomonas oryzae X11-5A  Xo_X11-5A_contig_110, whole genome shotgun  sequence >gi|332357382|gb|AFHK01000110.1| Xanthomonas oryzae X11-5A Xo_X11-5A_contig_110,  whole genome shotgun sequence  SEQ ID NO: 12 ATGAACCTGTCGAAAAAATCGCCAACCAAGGGTGCGGCATCGCTGCAGCG GTCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTGGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGGCGGGCCCGAGTACCAAGCAC GATCCGCACCATTGA >RaxX_Xoc_BSL256 A new nucleotide sequence  entered manually  SEQ ID NO: 13 ATGAACCTCTTGAAAAAATCGCCAACCAAGGGGGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTCGACCAGCGTCTGTGGA AACATGTCGGCGGTGGGGACTACCCCCCGTCGGGCCCGAATACCAAGCAC GATCCGCCGCCGCCCAACAACGGGCACCATTGA >RaxX_Xoc_MAI10 Xanthomonas oryzae pv. oryzicola  MAI10 172, whole genome shotgun sequence  SEQ ID NO: 14 ATGAACCTCTCGAAAAAATCGCCAACCAAGGGGGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTCGACCAGCGTCTGTGGA AACATGTCGGCGGTGGGGACTACCCCCCGTCGGGCCCGAATACCAAGCAC GATCCGCCGCCGCCCAACAACGGGCACCATTGA >RaxX_Xoo_AXO947 A new nucleotide sequence  entered manually  SEQ ID NO: 15 ATGAACCTCTCGAAAAAATCGCCAACCAAGGGTGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGTCCGACCGGAGCCGCTCGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTACCCCCCGTCGGGCCCGAATCCCAAGCAC GATCCGCCGCCGCGCAACAACGGGCACCATTGA >RaxX_Xoo_IXoo685 A new nucleotide sequence  entered manually  SEQ ID NO: 16 ATGAACCTCTCGAAAAAATCGCCAACCAAGGGTGCGGCATCGCTGCAGTG GCCCGCCAGGGCCAAGGGCCGACCGGAGCCGCTCGACCAGCGCCTGTGGA AGCATGTCGGCGGTGGGGACTACCCCCCGTCGGGCCCGAATACCAAGCAC GATCCGCCGCCGCCCAACAACGGGCACCATTGA >RaxX_Xoo_IXoo884 A new nucleotide sequence  entered manually  SEQ ID NO: 17 ATGAACCACTCGAAAAAATCGCCCACCAAGGGTGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGACCGGAGCAGCTCGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCGAATCCCAAGCAC GATCCGCCGCCGCGCAACCCCGGGCACCATTGA >RaxX_Xoo_NAI8 Xanthomonas oryzae pv. oryzae   NAI8 478, whole genome shotgun sequence SEQ ID NO: 18 ATGAACCTCTCGAAAAAATCGCCAACCAAGGGTGCGGCATCGCTGCAGCC GCCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTCGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTACCCCCCGTCGGGCCCGAATCCCAAGCAC GATCCGCCGCATCCGCCGCCGCGCAACAACGGGCACCATTGA >RaxX_Xoo_PXO99 Xanthomonas oryzae pv. oryzae  PXO99A chromosome, complete genome  SEQ ID NO: 19 ATGAACCACTCGAAAAAATCGCCCGCCAAGGGTGCGGCATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGACCGGAGCCGCTCGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCGAATCCCAAGCAC GATCCGCCGCCGCGCAACCCCGGGCACCATTGA >RaxX_Xsp._M97 Xanthomonas sp. M97 strain 97M  contig_1212, whole genome shotgun sequence  >gi|507136377|gb|AQPR01001212.1| Xanthomonas sp.  M97 contig_1212, whole genome shotgun sequence  SEQ ID NO: 20 ATGAACCTGTCGAAGAAATCGCCAACCTATGGCGCGCAATCGCTGCAGCG GCCCGCCGGGGCCAAGGGCCGGCTGGAGCCCCTGGACCAGCGTCTGTGGA AGCATGTCGGCGGTGGGGACTATCCCCCGCCGGGCGCGAACGACCGCCAC AACCCGCCACCGCCGCCGTCCAACGGCGGACACCACTGA >RaxX_Xvv_NCPPB206 A new nucleotide sequence  entered manually  SEQ ID NO: 21 ATGGAACTATTGAAAAAATCGCCATCCAATGGCGTGGGATCGCTCCAGCG GTCGGCCGGCGCCAAGGGCCGGCTAGAGCCGCTGGACCAGCGTCTGTGGA AGCATGTCGGGGGTGGGGACTATCCCCCACCGGGCTCGAATAACCGCCAC GATCCAACACAGCCGCCGCCCAAAAAAGGGAAACCTTGA 13 mer RaxX peptide  SEQ ID NO: 46 DYPPPGANPKHDP  

What is claimed is:
 1. An agricultural composition formulated for application to a plant comprising (a) a polypeptide comprising SEQ ID NO:46 or SEQ ID NO:70 and (b) a surfactant, herbicide, fungicide, pesticide, or fertilizer.
 2. The agricultural composition of claim 1 wherein the polypeptide comprises SEQ ID NO:70.
 3. The agricultural composition of claim 1 wherein the polypeptide comprises SEQ ID NO:46.
 4. The agricultural composition of claim 1, wherein the Tyr of SEQ ID NO:70 or SEQ ID NO:46 is sulfated.
 5. The agricultural formulation comprising the polypeptide of claim 1 and an agriculturally suitable carrier, surfactant, herbicide, fungicide, pesticide, or fertilizer.
 6. A method of enhancing root length in a plant, the method comprising contacting a plant with a sufficient amount of an agricultural composition formulated for application to a plant comprising a polypeptide comprising SEQ ID NO:46 or SEQ ID NO:70 to enhance root length of the plant compared to a plant that is not contacted by the polypeptide; wherein the contacting comprises spraying, atomizing, dusting or scattering the agricultural composition onto the plant or applying the agricultural composition to soil.
 7. The method of claim 6 wherein the polypeptide comprises SEQ ID NO:70.
 8. The method of claim 6 wherein the polypeptide comprises SEQ ID NO:46.
 9. The method of claim 6 wherein the contacting step comprises contacting the plant with a sufficient amount of the polypeptide comprises SEQ ID NO:46.
 10. The method of claim 6, wherein the plant is of a species selected from the group consisting of: Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, eucalyptus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Setaria, Miscanthus, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.
 11. A method of enhancing disease resistance in a plant, the method comprising contacting a plant with a sufficient amount of the agricultural composition formulated for application to a plant comprising a polypeptide comprising SEQ ID NO:46 or SEQ ID NO:70 to enhance disease resistance of the plant compared to a plant that is not contacted by the polypeptide, wherein the polypeptide comprises SEQ ID NO:70; wherein the contacting comprises spraying, atomizing, dusting or scattering the agricultural composition onto the plant or applying the agricultural composition to soil.
 12. The method of claim 11, wherein the plant is of a species selected from the group consisting of: Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, eucalyptus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Setaria, Miscanthus, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.
 13. The method of claim 6, wherein the Tyr of SEQ ID NO:70 or SEQ ID NO:46 is sulfated.
 14. The method of claim 6, wherein the agricultural composition comprises surfactant, herbicide, fungicide, pesticide, or fertilizer.
 15. The method of claim 11 wherein the polypeptide comprises SEQ ID NO:70.
 16. The method of claim 11 wherein the polypeptide comprises SEQ ID NO:46.
 17. The method of claim 11, wherein the Tyr of SEQ ID NO:70 or SEQ ID NO:46 is sulfated.
 18. The method of claim 11, wherein the agricultural composition comprises surfactant, herbicide, fungicide, pesticide, or fertilizer. 